Antibody-active agent conjugates and methods of use

ABSTRACT

The invention provides protein-active agent conjugates having an amino acid motif that can be recognized by an isoprenoid transferase. The invention also provides compositions containing the conjugates. The invention further provides methods for using the conjugates to deliver the active agent to a target cell, as well as methods for using the conjugates to treat a subject in need thereof (e.g., a subject in need of the active agent)

CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation of U.S. application Ser. No.14/517,616, filed Oct. 17, 2014 and now granted as U.S. Pat. No.9,669,107, which is a continuation of U.S. application Ser. No.14/181,648, filed on Feb. 15, 2014 and now abandoned, which is adivisional of U.S. application Ser. No. 13/466,875, filed on May 8, 2012and now abandoned, which claims the benefit of U.S. ProvisionalApplication No. 61/483,698, filed on May 8, 2011. The entire contents ofeach of the above-referenced applications are incorporated herein byreference in their entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Jun. 2, 2017, isnamed LCH-001.06_SL.txt and is 62,011 bytes in size.

BACKGROUND (a) Technical Field

The present disclosure relates to a protein-active agent conjugate. Theprotein (e.g., an oligopeptide, a polypeptide, an antibody, or the like)has substrate specificity for a desired target, and the active agent(e.g., a drug, a toxin, a ligand, a detection probe, and the like) has aspecific function or activity. The disclosure also relates to methodsfor preparing the conjugate. The disclosure further relates to methodsof using the conjugate to deliver an active agent to a target cell in asubject, as well as methods for treating a subject in need of the activeagent (e.g., a subject having cancer).

(b) Background Art

Methods for inhibiting growth of cancer cells by targeted delivery ofanti-cancer agents have been proposed. For example, it has been shownthat targeted delivery of an antibody-drug conjugate can kill aparticular cancer cell. As the antibody (or antibody fragment)specifically binds the cancer cell, the drug is delivered to the targetcancer cell. Targeted delivery of the drug ensures that the drug acts onthe target cancer cell instead of normal host cells, thereby minimizingthe side effects resulting from damage to normal cells.

Antibody conjugates can be used to deliver chemical and/or biologicalmolecules. Exemplary chemical and/or biological molecules include a drugconventionally used in chemical treatment, a bacterial protein toxin(e.g., diphtheria toxin), a plant protein toxin (e.g., ricin), a smallmolecule toxin (e.g., auristatin, geldanamycin, maytansinoid,calicheamycin, daunomycin, methotrexate, vindesine, and tubulysin), anaffinity ligand, a detection probe (e.g., fluorescent probe, radioactiveprobe), and the like (including combinations thereof).

Antibody-drug conjugates that have been proposed thus far are preparedby bonding a drug moiety with a plurality of lysine groups of anantibody. Alternatively, antibody-drug conjugates are prepared byreducing all or part of the interchain disulfide groups of an antibodyor reducing all the interchain disulfide groups followed by partialoxidation to thereby give free cysteine thiol groups, and then bondingthe free cysteine thiol groups with a drug moiety.

Existing preparation methods, however, have some problems. For example,the overall preparation process is complicated because the antibody-drugconjugates prepared by the existing preparation methods are not uniform(homogeneous). When antibody-drug conjugates are prepared by bonding adrug moiety with lysine groups, various types and forms of antibody-drugconjugates are obtained due to the presence of many lysine groups in theantibody (e.g., 100 lysine groups per antibody). Similarly, whenpreparing antibody-drug conjugates by bonding thiol groups with a drugmoiety, a mixture of diastereomers is obtained due to bonding betweenthiol groups and maleimide groups. For example, if n drugs areconjugated, a mixture of 2^(n) stereoisomers is obtained. Thus, wherethe drug distribution number is 0-8 (e.g., where interchain disulfidegroups are reduced), a mixture of

$\sum\limits_{n = 0}^{n = 8}2^{n}$

of stereoisomers is obtained. In addition, where i drugs are conjugatedwith q sites, a mixture of

$\sum\limits_{i = 0}^{q}{qCi}$

of different compounds is obtained.

Furthermore, when preparing antibody-drug conjugates by bonding lysinegroups with a drug moiety, the electric charge of the lysine groups maybe lost, thereby causing the antibody to lose its unique antigenspecificity. Likewise, the tertiary or quaternary structure of theantibody may not be maintained when preparing antibody-drug conjugatesby reducing disulfide groups, thereby causing the antibody to beinactivated or become a non-specific antibody. When preparingantibody-drug conjugates by using thiol-maleimide bonding, the drug maybe cleaved (non-specifically) from the conjugates via, e.g., a reversereaction.

To overcome the problems associated with the prior preparation methods,an alternative method was proposed in which amino acid groups inparticular positions of an antibody are replaced with cysteine groups.Although this method shows better result than the prior preparationmethods in terms of toxicity, activity, and safety, this method stillinvolves thiol-maleimide bonding and thus suffers from the diastereomerand instability problems associated with thiol-maleimide bonding.Another alternative method was proposed in which selenocysteine groupsare attached to the carboxy terminals of an antibody.

In addition to use of cysteine substitutions to control the site ofconjugation, Ambrx Technology (at the World Wide Web (www) ambrx.com)has been working toward incorporating non-natural amino acids in theantibody to provide functional groups that can be used for linkerchemistry. Ambrx's expression systems contain tRNA synthetases thataminoacylate the original tRNA with a non-natural amino acid, therebyinserting a non-natural amino acid whenever the amber stop isencountered.

Redwood Bioscience's (at the World Wide Web (www) redwoodbioscience.com)technology employs genetically encoded aldehyde tags and aims to exploita specific sequence that is posttranslationally recognized and modifiedby an enzyme, i.e., a formyl glycine-generating enzyme, to produce aso-called aldehyde chemical handle. The incorporation of a CxPxRsequence at specific positions in the antibody provides a means toproduce a reactive aldehyde amenable to drug conjugation.

However, in view of the above-mentioned problems in the art pertainingto making antibody-drug conjugates, new antibody-drug conjugates and newmethods of making antibody-drug conjugates are highly desirable.

The above information disclosed in this Background section is only forenhancement of understanding of the background of the invention andtherefore it may contain information that does not form the prior artthat is already known to a person of ordinary skill in the art.

SUMMARY OF THE DISCLOSURE

As described below, the present invention generally featuresprotein-active agent conjugates and methods for making theprotein-active agent conjugates. The invention also features methods fordelivering the protein-active agent conjugate to a target cell in asubject, as well as methods for treating a subject in need of the activeagent. The protein-active agent conjugates of the invention can beproduced homogeneously and advantageously used for targeted treatment ofa disease.

In aspects, the invention provides protein-active agent conjugates. Inembodiments, the protein has an amino acid motif that can be recognizedby an isoprenoid transferase. In embodiments, the active agent iscovalently linked to the protein at the amino acid motif.

In embodiments, the protein has a deletion in the carboxy terminus ofthe protein. In related embodiments, the modification is attached to theamino acid motif.

In embodiments, the protein has an oligopeptide or polypeptide additionin the carboxy terminus of the protein. In related embodiments, themodification is attached to the amino acid motif.

In embodiments, the protein has a deletion in the carboxy terminus ofthe protein and an oligopeptide or polypeptide addition in the carboxyterminus of the protein. In related embodiments, the modification isattached to the amino acid motif.

In embodiments, the protein is an antibody or a fragment of an antigenicpolypeptide. In related embodiments, the protein is a monoclonalantibody. In related embodiments, at least one light chain and/or atleast one heavy chain of the monoclonal antibody comprises an amino acidregion having the amino acid motif.

In any of the above aspects or embodiments, the isoprenoid transferaseis FTase or GGTase.

In any of the above aspects or embodiments, the active agent is a drug,a toxin, an affinity ligand, a detection probe, or a combinationthereof.

In any of the above aspects or embodiments, the amino acid motif isCAAX, XXCC, XCXC, or CXX, wherein C represents cysteine, A represents analiphatic amino acid, and X represents an amino acid that determines asubstrate specificity of the isoprenoid transferase.

In any of the above aspects or embodiments, the amino acid motif iscovalently linked to the active agent via at least one linker. Inrelated embodiments, the linker is an isoprenyl derivative that can berecognized by the isoprenoid transferase.

In related embodiments, the linker is represented by the followingformula (I):

wherein,

P₁ and Y is independently a group containing a first functional group(FG1), the FG1 being selected from the group consisting of: acetylene,azide, aldehyde, hydroxylamine, hydrazine, ketone, nitrobenzofurazan(NBD), dansyl, fluorescein, biotin, and Rhodamine,

L₁ is (CH₂)_(r)X_(q)(CH₂)_(p),

X is oxygen, sulfur, —NR₁—, —C(O)NR₁—, —NR₁C(O)—, —NR₁SO₂—, —SO₂NR₁—,—(CH═CH)—, or acetylene,

R₁ is hydrogen, C₁₋₆ alkyl, C₁₋₆ alkyl aryl, or C₁₋₆ alkyl heteroaryl,

r and p is independently an integer of 0 to 6,

q is an integer of 0 to 1, and

n is an integer of 1 to 4.

In embodiments, the active agent is attached to a group containing asecond functional group (FG2) that can react with the FG1. In relatedembodiments, FG2 is an acetylene, hydroxylamine, azide, aldehyde,hydrazine, ketone, or amine. In further related embodiments, the activeagent is attached to the group containing an FG2 via—(CH₂)_(r)X_(q)(CH₂)_(p)— or —[ZCH₂CH₂O(CH₂CH₂O)_(w)CH₂CH₂Z]—, in which

X is oxygen, sulfur, —NR₁—, —C(O)NR₁—, —NR₁C(O)—, —NRISO₂—, or —SO₂NR₁—,

Z is oxygen, sulfur or NR₁,

R₁ is hydrogen, C₁₋₆ alkyl, C₁₋₆ alkyl aryl, or C₁₋₆ alkyl heteroaryl,

r and p is independently an integer of 0 to 6,

q is an integer of 0 to 1, and

m is an integer of 0 to 6.

In yet further related embodiments, the —(CH₂)_(r)X_(q)(CH₂)_(p)— or—[ZCH₂CH₂O(CH₂CH₂O)_(w)CH₂CH₂Z]— is attached to (i) a peptide(s) thatcan be cleaved by cathepsin B or (ii) a glucuronide that can be cleavedby β-glucuronidase.

In embodiments, the peptide that can be cleaved by cathepsin B is

In embodiments, the glucuronide that can be cleaved by β-glucuronidaseis

In aspects, the invention provides methods for preparing any of theprotein-active agent conjugate described herein. In embodiments, themethods involve expressing a protein having an amino acid motif that canbe recognized by an isoprenoid transferase. In embodiments, the methodsinvolve enzymatically reacting, with the isoprenoid transferase, theexpressed protein and at least one isosubstrate having a firstfunctional group (FG1), thereby producing a functionalized protein. Inembodiments, the methods involve attaching a second functional group(FG2) to an active agent, thereby producing a functionalized activeagent. In embodiments, the methods involve reacting the functionalizedprotein with the functionalized active agent, thereby producing theprotein-active agent conjugate.

In related embodiments, the amino acid motif is in the carboxy terminusof the protein.

In related embodiments, the amino acid motif is CAAX, XXCC, XCXC, orCXX, wherein C represents cysteine, A represents an aliphatic aminoacid, and X represents an amino acid that determines the substratespecificity of the isoprenoid transferase.

In related embodiments, the amino acid motif is CAAX, and wherein themethod further comprises removing AAX from the amino acid motif afterstep (b).

In related embodiments, the FG2 is attached to the active agent by atleast one linker.

In related embodiments, the reaction between the functionalized proteinand the functionalized active agent is click chemistry reaction or ahydrazone and/or oxime formation. In embodiments, the FG1 is an azidegroup and the FG2 is an acetylene group. In embodiments, the FG1 is anacetylene group and the FG2 is an azide group. In embodiments, the FG1is an aldehyde or ketone group and the FG2 is a hydrazine orhydroxylamine. In embodiments, the FG1 is hydrazine or hydroxylamine andthe FG2 is an aldehyde or ketone.

In aspects, the invention provides methods for preparing any of theprotein-active agent conjugate described herein, and the methods involveexpressing a protein having an amino acid motif that can be recognizedby an isoprenoid transferase. In embodiments, the methods involveattaching an isosubstrate of an isoprenoid transferase to an activeagent. In embodiments, the methods involve enzymatically reacting, withthe isoprenoid transferase, the expressed protein and the active agentattached to the isosubstrate.

In related embodiments, the amino acid motif is in the carboxy terminusof the protein.

In related embodiments, the amino acid motif is CAAX, XXCC, XCXC, orCXX, wherein C represents cysteine, A represents an aliphatic aminoacid, and X represents an amino acid that determines the substratespecificity of the isoprenoid transferase.

In related embodiments, the isosubstrate is attached to the active agentby at least one linker.

In aspects, the invention provides compositions containing any of theprotein-active agent conjugates described herein. In embodiments, thecomposition is a homogeneous mixture of the protein-active agentconjugate. In embodiments, the protein is an antibody or a fragment ofan antigenic polypeptide.

In aspects, the invention provides methods for delivering an activeagent to a target cell in a subject. In embodiments, the methods involveadministering at least one of the protein-active agent conjugates orcompositions described herein. In embodiments, the target cell is acancer cell.

In aspects, the invention provides methods for treating a subject inneed thereof (i.e., in need of the active agent). In embodiments, themethods involve administering at least one of the protein-active agentconjugates or compositions described herein. In embodiments, the subjecthas cancer. In embodiments, the subject has an infection with apathogenic agent. The pathogenic agent may be a virus, bacteria, fungus,or parasite.

In the above-described protein-active agent conjugates, compositions,and methods, in some embodiments, the active agent may be animmunomodulatory compound, an anti-cancer agent, an anti-viral agent, ananti-bacterial agent, an anti-fungal agent, or an anti-parasitic agent.

The above and other aspects, features, and advantages of the presentinvention will be apparent from or are set forth in more detail in theaccompanying drawings, which are incorporated in and form a part of thisspecification, and the following Detailed Description, which togetherserve to explain by way of example the principles of the presentinvention.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows an amino acid sequence of a modified Herceptin antibody(Herceptin-HC-GCVIM) (“Herceptin-HC-GCVIM” disclosed as SEQ ID NO: 8)prepared by inserting GCVIM (SEQ ID NO: 1) to the C-terminus of theheavy chain of Herceptin. Figure discloses SEQ ID NOS 8-9, respectively,in order of appearance.

FIG. 2 shows an amino acid sequence of a modified Herceptin antibody(Herceptin-LC-GCVIM) (“Herceptin-LC-GCVIM” disclosed as SEQ ID NO: 11)prepared by inserting GCVIM (SEQ ID NO: 1) to the C-terminus of thelight chain of Herceptin. Figure discloses SEQ ID NOS 10-11,respectively, in order of appearance.

FIG. 3 shows an amino acid sequence of a modified Herceptin antibody(Herceptin-HC-G₅CVIM) (“Herceptin-HC-G₅CVIM” disclosed as SEQ ID NO: 12)prepared by inserting G₅CVIM (SEQ ID NO: 2) to the C-terminus of theheavy chain of Herceptin. Figure discloses SEQ ID NOS 12-13,respectively, in order of appearance.

FIG. 4 shows an amino acid sequence of a modified Herceptin antibody(Herceptin-LC-G₅CVIM) (“Herceptin-LC-G₅CVIM” disclosed as SEQ ID NO: 15)prepared by inserting G₅CVIM (SEQ ID NO: 2) to the C-terminus of thelight chain of Herceptin. Figure discloses SEQ ID NOS 14-15,respectively, in order of appearance.

FIG. 5 shows an amino acid sequence of a modified Herceptin antibody(Herceptin-HC-G₇CVIM) (“Herceptin-HC-G₇CVIM” disclosed as SEQ ID NO: 16)prepared by inserting G₇CVIM (SEQ ID NO: 3) to the C-terminus of theheavy chain of Herceptin. Figure discloses SEQ ID NOS 16-17,respectively, in order of appearance.

FIG. 6 shows an amino acid sequence of a modified Herceptin antibody(Herceptin-LC-G₇CVIM) (“Herceptin-LC-G₇CVIM” disclosed as SEQ ID NO: 19)prepared by inserting G₇CVIM (SEQ ID NO: 3) to the C-terminus of thelight chain of Herceptin. Figure discloses SEQ ID NOS 18-19,respectively, in order of appearance.

FIG. 7 shows an amino acid sequence of a modified Herceptin antibody(Herceptin-HC-G₁₀CVIM) (“Herceptin-HC-G₁₀CVIM” disclosed as SEQ ID NO:20) prepared by inserting G₁₀CVIM (SEQ ID NO: 4) to the C-terminus ofthe heavy chain of Herceptin. Figure discloses SEQ ID NOS 20-21,respectively, in order of appearance.

FIG. 8 shows an amino acid sequence of a modified Herceptin antibody(Herceptin-LC-G₁₀CVIM) (“Herceptin-LC-G₁₀CVIM” disclosed as SEQ ID NO:23) prepared by inserting G₁₀CVIM (SEQ ID NO: 4) to the C-terminus ofthe light chain of Herceptin. Figure discloses SEQ ID NOS 22-23,respectively, in order of appearance.

FIG. 9 shows an amino acid sequence of a modified Herceptin antibody(Herceptin-HC-G₁₀CVLL) (“Herceptin-HC-G₁₀CVLL” disclosed as SEQ ID NO:24) prepared by inserting G₁₀CVLL (SEQ ID NO: 6) to the C-terminus ofthe heavy chain of Herceptin. Figure discloses SEQ ID NOS 24-25,respectively, in order of appearance.

FIG. 10 shows an amino acid sequence of a modified Herceptin antibody(Herceptin-LC-G₁₀CVLL) (“Herceptin-LC-G₁₀CVLL” disclosed as SEQ ID NO:27) prepared by inserting G₁₀CVLL (SEQ ID NO: 6) to the C-terminus ofthe light chain of Herceptin. Figure discloses SEQ ID NOS 26-27,respectively, in order of appearance.

FIG. 11 shows an SDS-PAGE gel analyzing a modified anti cMET antibody(anti cMET-HC-G₇CVIM) (“G₇CVIM” disclosed as SEQ ID NO: 3) prepared byinserting G₇CVIM (SEQ ID NO: 3) to the C-terminus of the heavy chain ofanti cMET antibody, a modified anti cMET antibody (anti cMET-LC-G₇CVIM)(“G₇CVIM” disclosed as SEQ ID NO: 3) prepared by inserting G₇CVIM (SEQID NO: 3) to the C-terminus of the light chain of anti cMET antibody, amodified anti cMET antibody (anti cMET-HC-G₁₀CVIM) (“G₁₀CVIM” disclosedas SEQ ID NO: 4) prepared by inserting G₁₀CVIM (SEQ ID NO: 4) to theC-terminus of the heavy chain of anti cMET antibody, and a modified anticMET antibody (anti cMET-LC-G₁₀CVIM) (“G₁₀CVIM” disclosed as SEQ ID NO:4) prepared by inserting G₁₀CVIM (SEQ ID NO: 4) to the C-terminus of thelight chain of anti cMET antibody.

FIG. 12 shows an SDS-PAGE gel analyzing prenylation ofHerceptin-HC-G_(n)CVIM (“G_(n)CVIM” disclosed as SEQ ID NO: 5) by usingFTase and NBD-GPP. Figure discloses “G₇CVIM” as SEQ ID NO: 3 and“G₁₀CVIM” as SEQ ID NO: 4.

FIG. 13 shows an SDS-PAGE gel analyzing prenylation ofHerceptin-LC-G_(n)CVIM (“G_(n)CVIM” disclosed as SEQ ID NO: 5) by usingFTase and NBD-GPP. Figure discloses “G₅CVIM” as SEQ ID NO: 2, “G₇CVIM”as SEQ ID NO: 3, and “G₁₀CVIM” as SEQ ID NO: 4.

FIG. 14 shows an SDS-PAGE gel analyzing prenylation of cMET-HC-G_(n)CVIM(“G_(n)CVIM” disclosed as SEQ ID NO: 5) by using FTase and NBD-GPP.Figure discloses “G₇CVIM” as SEQ ID NO: 3 and “G₁₀CVIM” as SEQ ID NO: 4.

FIG. 15 shows an SDS-PAGE gel analyzing prenylation of cMET-LC-G_(n)CVIM(“G_(n)CVIM” disclosed as SEQ ID NO: 5) by using FTase and NBD-GPP.Figure discloses “G₇CVIM” as SEQ ID NO: 3 and “G₁₀CVIM” as SEQ ID NO: 4.

FIG. 16 shows an SDS-PAGE gel analyzing prenylation ofHerceptin-HC-G₁₀CVLL (“Herceptin-HC-G₁₀CVLL” disclosed as SEQ ID NO: 24)and Herceptin-LC-G₁₀CVLL (“Herceptin-LC-G₁₀CVLL” disclosed as SEQ ID NO:27) by using FTase/NBD-GPP or GGTase I/NBD-FPP. Figure discloses“G₇CVIM” as SEQ ID NO: 3 and “G₁₀CVLL” as SEQ ID NO: 6.

FIG. 17 shows the results from LC/MS analysis of a prenylatedHerceptin-LC-GCVIM (“Herceptin-LC-G₇CVIM” disclosed as SEQ ID NO: 19).

FIG. 18 shows the results from LC/MS analysis of a prenylatedHerceptin-LC-G₁₀CVIM (“Herceptin-LC-G₁₀CVIM” disclosed as SEQ ID NO:23).

FIG. 19 shows the results from LC/MS and deconvoluted mass spectraanalysis of LCB14-0104 (Herceptin-LC-G₇CVIM-NC-MMAF-Ome) (“G₇CVIM”disclosed as SEQ ID NO: 3).

FIG. 20 shows the HIC-HPLC chromatograms of Herceptin-LC-G₇CVIM(“Herceptin-LC-G₇CVIM” disclosed as SEQ ID NO: 19), prenylatedHerceptin-LC-G₇CVIM (“Herceptin-LC-G₇CVIM” disclosed as SEQ ID NO: 19),and LCB14-0101 (Herceptin-LC-G₇CVIM-BG-MMAF) (“G₇CVIM” disclosed as SEQID NO: 3).

FIG. 21 shows the results from an anti-proliferation assay of LCB14-0101(Herceptin-LC-G₇CVIM-BG-MMAF) (“G₇CVIM” disclosed as SEQ ID NO: 3) withbreast cancer cell lines MCF-7, MDA-MB-468, and SK-BR-3.

FIG. 22 shows the results from an anti-proliferation assay of LCB14-0102(Herceptin-LC-G₇CVIM-VC-MMAF-OMe) (“G₇CVIM” disclosed as SEQ ID NO: 3)with breast cancer cell lines MCF-7 and SK-BR-3.

FIG. 23 shows the results from an anti-proliferation assay of LCB14-0103(Herceptin-LC-G₇CVIM-BG-MMAE) (“G₇CVIM” disclosed as SEQ ID NO: 3) withbreast cancer cell lines MCF-7 and SK-BR-3.

FIG. 24 shows a process of posttranslational modification of a protein(C-terminal CVIM (SEQ ID NO: 28)).

FIG. 25 shows a mechanism of release of active drugs (exceptnon-cleavable linker).

FIG. 26 shows the chemical structures of antibody-drug conjugatesLCB14-0101, LCB14-0102, LCB14-0103, and LCB14-0104. Figure discloses“CVIM” as SEQ ID NO: 28.

FIG. 27 is a schematic diagram depicting a process for preparing aprotein-active agent conjugate by using an isoprenoid transferase and anisosubstrate thereof in which cysteine of the CAAX motif is alkylated.

DETAILED DESCRIPTION OF THE DISCLOSURE

Reference will now be made in detail to embodiments of the presentinvention, examples of which are illustrated in the drawings attachedhereinafter, wherein like reference numerals refer to like elementsthroughout. The embodiments are described below so as to explain thepresent invention by referring to the figures.

Definitions

By “agent” or “active agent” is meant any small molecule chemicalcompound, antibody, nucleic acid molecule, or polypeptide, or fragmentsthereof. Examples include, but are not limited to, a drug, a toxin, anaffinity ligand, a detection probe, or a combination thereof.

By “analog” is meant a molecule that is not identical, but has analogousfunctional or structural features. For example, a polypeptide analogretains the biological activity of a corresponding naturally-occurringpolypeptide, while having certain biochemical modifications that enhancethe analog's function relative to a naturally occurring polypeptide.Such biochemical modifications could increase the analog's proteaseresistance, membrane permeability, or half-life, without altering, forexample, ligand binding. An analog may include an unnatural amino acid.

In this disclosure, “comprises,” “comprising,” “containing” and “having”and the like can have the meaning ascribed to them in U.S. Patent lawand can mean “includes,” “including,” and the like; “consistingessentially of” or “consists essentially” likewise has the meaningascribed in U.S. Patent law and the term is open-ended, allowing for thepresence of more than that which is recited so long as basic or novelcharacteristics of that which is recited is not changed by the presenceof more than that which is recited, but excludes prior art embodiments.

“Contacting a cell” is understood herein as providing an agent to a celle.g., a cell to be treated in culture, ex vivo, or in an animal, suchthat the agent can interact with the cell (e.g., cell to be treated),potentially be taken up by the cell, and have an effect on the cell. Theagent (e.g., an adjuvant) can be delivered to the cell directly (e.g.,by addition of the agent to culture medium or by injection into the cellor tissue of interest), or by delivery to the organism by a topical orparenteral route of administration for delivery to the cell by vascular,lymphatic, or other means. One of ordinary skill in the art will readilyunderstand that administration of the protein-active agent conjugates ofthe invention to a subject involves contacting the protein-active agentconjugate with a cell of the subject.

By “disease” is meant any condition or disorder that damages orinterferes with the normal function of a cell, tissue, or organ.

The terms “effective amount,” “therapeutically effective amount,”“effective dose,” or “therapeutically effective dose” refers to thatamount of an agent to produce the intended pharmacological, therapeutic,or preventive result. For example, the pharmacologically effectiveamount results in the prevention or delay of onset of disease, either inan individual or in the frequency of disease in a population. More thanone dose may be required to provide an effective dose. It is understoodthat an effective dose in one population may or may not be sufficient inall populations. Thus, in connection with the administration of an agentor immunogenic composition, the agent or immunogenic composition is“effective against” a disease or condition when administration in aclinically appropriate manner results in a beneficial effect for atleast a statistically significant fraction of subjects, such as aprevention of disease onset, improvement of symptoms, a cure, areduction in disease signs or symptoms, extension of life, improvementin quality of life, or other effect generally recognized as positive bymedical doctors familiar with treating the particular type of disease orcondition.

By “enhances” is meant a positive alteration of at least 10%, 25%, 50%,75%, 100%, or any number therebetween.

By “fragment” is meant a portion of a polypeptide or nucleic acidmolecule. This portion contains, preferably, at least 10%, 20%, 30%,40%, 50%, 60%, 70%, 80%, or 90% of the entire length of the referencenucleic acid molecule or polypeptide. A fragment may contain 10, 20, 30,40, 50, 60, 70, 80, 90, or 100, 200, 300, 400, 500, 600, 700, 800, 900,or 1000 nucleotides or amino acids.

“Hybridization” means hydrogen bonding, which may be Watson-Crick,Hoogsteen or reversed Hoogsteen hydrogen bonding, between complementarynucleotide bases. For example, adenine and thymine are complementarynucleotide bases that pair through the formation of hydrogen bonds.

“Obtaining” is understood herein as manufacturing, purchasing,synthesizing, isolating, purifying, or otherwise coming into possessionof.

The phrase “pharmaceutically acceptable carrier, excipient, or diluent”is art recognized and includes a pharmaceutically acceptable material,composition or vehicle, suitable for administering compounds of thepresent invention to mammals. As used herein, the term “pharmaceuticallyacceptable” means being approved by a regulatory agency of the Federalor a state government or listed in the U.S. Pharmacopia, EuropeanPharmacopia or other generally recognized pharmacopia for use inmammals, e.g., humans.

By “reduces” is meant a negative alteration of at least 10%, 25%, 50%,75%, 100%, or any number therebetween.

By “reference” is meant a standard or control condition.

A “sample” as used herein refers to a biological material that isisolated from its environment (e.g., blood or tissue from an animal,cells, or conditioned media from tissue culture). In embodiments, thesample is suspected of containing, or known to contain an analyte, suchas a protein of interest (e.g., antibody, cytokine, and the like). Asample can also be a partially purified fraction of a tissue or bodilyfluid. A reference sample can be a “normal” sample, from a donor nothaving the disease or condition fluid, or from a normal tissue in asubject having the disease or condition, or an untreated subject (e.g.,a subject not treated with the vaccine). A reference sample can also betaken at a “zero time point” prior to contacting the cell or subjectwith the agent or therapeutic intervention to be tested.

By “specifically binds” is meant recognition and binding to a target(e.g., polypeptide, cell, and the like), but which does notsubstantially recognize and bind other molecules in a sample, forexample, a biological sample.

A “subject” as used herein refers to a living organism. In embodiments,the living organism is an animal. In embodiments, the subject is amammal. In embodiments, the subject is a domesticated mammal or aprimate including a non-human primate. Examples of subjects include, butare not limited to, humans, monkeys, dogs, cats, mice, rats, cows,horses, swine, goats, sheep, and birds. A subject may also be referredto as a patient.

A subject “suffering from or suspected of suffering from” a specificdisease, condition, or syndrome has a sufficient number of risk factorsor presents with a sufficient number or combination of signs or symptomsof the disease, condition, or syndrome such that a competent individualwould diagnose or suspect that the subject was suffering from thedisease, condition, or syndrome. Methods for identification of subjectssuffering from or suspected of suffering from a disease or condition iswithin the ability of those in the art. Subjects suffering from, andsuspected of suffering from, a specific disease, condition, or syndromeare not necessarily two distinct groups. One of ordinary skill in theart would also readily understand that a subject in need of an activeagent may also be a subject suffering from or suspected of sufferingfrom a specific disease, condition, or syndrome.

As used herein, the terms “treat,” “treating,” “treatment,” and the likerefer to reducing or ameliorating a disorder and/or symptoms associatedtherewith (e.g., cancer or cancer associated symptoms). It will beappreciated that, although not precluded, treating a disorder orcondition does not require that the disorder, condition or symptomsassociated therewith be completely eliminated.

Ranges provided herein are understood to be shorthand for all of thevalues within the range. For example, a range of 1 to 50 is understoodto include any number, combination of numbers, or sub-range from thegroup consisting 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50.

Unless specifically stated or obvious from context, as used herein, theterm “or” is understood to be inclusive.

Unless specifically stated or obvious from context, as used herein, theterms “a”, “an”, and “the” are understood to be singular or plural.

Unless specifically stated or obvious from context, as used herein, theterm “about” is understood as within a range of normal tolerance in theart, for example within 2 standard deviations of the mean. About can beunderstood as within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%,0.1%, 0.05%, or 0.01% of the stated value. Unless otherwise clear fromcontext, all numerical values provided herein can be modified by theterm about.

The recitation of a listing of chemical groups in any definition of avariable herein includes definitions of that variable as any singlegroup or combination of listed groups. The recitation of an embodimentfor a variable or aspect herein includes that embodiment as any singleembodiment or in combination with any other embodiments or portionsthereof.

Any compositions or methods provided herein can be combined with one ormore of any of the other compositions and methods provided herein.

1. Methods for Preparing Protein-Active Agent Conjugates

Methods for making the protein-active agent conjugates of the inventionand variations thereof are readily apparent to one of ordinary skill inthe art based on the disclosures herein. Provided below are exemplarymethods, which are provided by way of illustration, and are not intendedto be limiting of the present invention.

Embodiment 1

A method for preparing a protein-active agent conjugate according to oneembodiment of the invention comprises: (a) expressing a protein havingan amino acid motif that can be recognized by an isoprenoid transferase;(b) enzymatically reacting, using the isoprenoid transferase, theexpressed protein and at least one isosubstrate having a firstfunctional group (FG1), thereby producing a functionalized protein; (c)attaching a second functional group (FG2) to an active agent, therebyproducing a functionalized active agent; and (d) reacting thefunctionalized protein with the functionalized active agent, therebyproducing the protein-active agent conjugate.

The term “protein” used herein is understood as two or moreindependently selected natural or non-natural amino acids joined by acovalent bond (e.g., a peptide bond). A peptide can include 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more naturalor non-natural amino acids joined by peptide bonds. Polypeptides asdescribed herein include full length proteins (e.g., fully processedproteins) as well as shorter amino acids sequences (e.g., fragments ofnaturally occurring proteins or synthetic polypeptide fragments).

A protein refers to an oligopeptide or polypeptide containing at leastone C-terminus and at least one N-terminus. The term is used herein toinclude an intact oligopeptide or polypeptide, a modified form thereof,a fragment thereof, and analogs thereof. For example, the term can referto an oligopeptide or polypeptide, or an oligopeptide or polypeptidemodified by attaching thereto an amino acid sequence that can berecognized by an isoprenoid transferase. The term “fragment” used hereinrefers to a portion of the amino acid sequence consisting of anoligopeptide or polypeptide. The term is used herein to include aportion of the amino acid sequence that has the substrate specificity ofthe oligopeptide or polypeptide. The term “analog” refers to anoligopeptide or polypeptide having a sequence identity of at least 70%or 75%, at least 80% or 85%, at least 90%, 91%, 92%, 93%, 94%, or 95%,or at least 96, 97%, 98%, or 9% with a reference oligopeptide orpolypeptide.

The term “protein” used herein also includes an antibody a fragment ofan antigenic polypeptide, or an analog or derivative thereof. The term“antibody” means an immunoglobulin molecule that recognizes andspecifically binds to a target, such as a protein, polypeptide, peptide,carbohydrate, polynucleotide, lipid, or combinations of the foregoingthrough at least one antigen recognition site within the variable regionof the immunoglobulin molecule. As used herein, the term “antibody”encompasses intact polyclonal antibodies, intact monoclonal antibodies,antibody fragments (such as Fab, Fab′, F(ab′)₂, Fd, and Fv fragments),single chain Fv (scFv) mutants, multispecific antibodies such asbispecific antibodies generated from at least two intact antibodies,chimeric antibodies, humanized antibodies, human antibodies, fusionproteins comprising an antigen determination portion of an antibody, andany other modified immunoglobulin molecule comprising an antigenrecognition site so long as the antibodies exhibit the desiredbiological activity. An antibody can be of any the five major classes ofimmunoglobulins: IgA, IgD, IgE, IgG, and IgM, or subclasses (isotypes)thereof (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2), based on theidentity of their heavy-chain constant domains referred to as alpha,delta, epsilon, gamma, and mu, respectively. The different classes ofimmunoglobulins have different and well known subunit structures andthree-dimensional configurations.

The term “antibody fragment” refers to a portion of an intact antibodyand refers to the antigenic determining variable regions of an intactantibody. Examples of antibody fragments include, but are not limited toFab, Fab′, F(ab′)₂, Fd, and Fv fragments, linear antibodies, singlechain antibodies, and multispecific antibodies formed from antibodyfragments.

A “monoclonal antibody” refers to homogenous antibody populationinvolved in the highly specific recognition and binding of a singleantigenic determinant, or epitope. This is in contrast to polyclonalantibodies that typically include different antibodies directed againstdifferent antigenic determinants. The term “monoclonal antibody”encompasses both intact and full-length monoclonal antibodies as well asantibody fragments (such as Fab, Fab′, F(ab′)₂, Fd, Fv), single chain(scFv) mutants, fusion proteins comprising an antibody portion, and anyother modified immunoglobulin molecule comprising an antigen recognitionsite. Furthermore, “monoclonal antibody” refers to such antibodies madein any number of manners including but not limited to by hybridoma,phage selection, recombinant expression, and transgenic animals.

The term “humanized antibody” refers to forms of non-human (e.g.,murine) antibodies that are specific immunoglobulin chains, chimericimmunoglobulins, or fragments thereof that contain minimal non-human(e.g., murine) sequences. Typically, humanized antibodies are humanimmunoglobulins in which residues from the complementary determiningregion (CDR) are replaced by residues from the CDR of a non-humanspecies (e.g., mouse, rat, rabbit, and hamster) that have the desiredspecificity, affinity, and capability (Jones et al., 1986, Nature,321:522-525; Riechmann et al., 1988, Nature, 332:323-327; Verhoeyen etal., 1988, Science, 239:1534-1536). In some instances, the Fv frameworkregion (FR) residues of a human immunoglobulin are replaced with thecorresponding residues in an antibody from a non-human species that hasthe desired specificity, affinity, and capability. The humanizedantibody can be further modified by the substitution of additionalresidue either in the Fv framework region and/or within the replacednon-human residues to refine and optimize antibody specificity,affinity, and/or capability. In general, the humanized antibody willcomprise substantially all of at least one, and typically two or three,variable domains containing all or substantially all of the CDR regionsthat correspond to the non-human immunoglobulin whereas all orsubstantially all of the FR regions are those of a human immunoglobulinconsensus sequence. The humanized antibody can also comprise at least aportion of an immunoglobulin constant region or domain (Fc), typicallythat of a human immunoglobulin. Examples of methods used to generatehumanized antibodies are described in U.S. Pat. No. 5,225,539.

The term “human antibody” means an antibody produced by a human or anantibody having an amino acid sequence corresponding to an antibodyproduced by a human made using any technique known in the art. Thisdefinition of a human antibody includes intact or full-lengthantibodies, fragments thereof, and/or antibodies comprising at least onehuman heavy and/or light chain polypeptide such as, for example, anantibody comprising murine light chain and human heavy chainpolypeptides.

The term “chimeric antibodies” refers to antibodies wherein the aminoacid sequence of the immunoglobulin molecule is derived from two or morespecies. Typically, the variable region of both light and heavy chainscorresponds to the variable region of antibodies derived from onespecies of mammals (e.g., mouse, rat, rabbit, etc) with the desiredspecificity, affinity, and capability while the constant regions arehomologous to the sequences in antibodies derived from another (usuallyhuman) to avoid eliciting an immune response in that species.

The term “epitope” or “antigenic determinant” are used interchangeablyherein and refer to that portion of an antigen capable of beingrecognized and specifically bound by a particular antibody. When theantigen is a polypeptide, epitopes can be formed both from contiguousamino acids and noncontiguous amino acids juxtaposed by tertiary foldingof a protein. Epitopes formed from contiguous amino acids are typicallyretained upon protein denaturing, whereas epitopes formed by tertiaryfolding are typically lost upon protein denaturing. An epitope typicallyincludes at least 3, at least 5, or at least 8-10 amino acids in aunique spatial conformation.

That an antibody “specifically binds” to an epitope or antigenicmolecule means that the antibody reacts or associates more frequently,more rapidly, with greater duration, with greater affinity, or with somecombination of the above to an epitope or antigenic molecule than withalternative substances, including unrelated proteins. In certainembodiments, “specifically binds” means, for instance, that an antibodybinds to a protein with a K_(D) of about 0.1 mM or less, but moreusually less than about 1 μM. In certain embodiments, “specificallybinds” means that an antibody binds to a protein at times with a K_(D)of at least about 0.1 μM or less, and at other times at least about 0.01μM or less. Because of the sequence identity between homologous proteinsin different species, specific binding can include an antibody thatrecognizes a particular protein in more than one species. It isunderstood that an antibody or binding moiety that specifically binds toa first target may or may not specifically bind to a second target. Assuch, “specific binding” does not necessarily require (although it caninclude) exclusive binding, i.e. binding to a single target. Generally,but not necessarily, reference to binding means specific binding.

The antibodies, including fragments/derivatives thereof and monoclonalantibodies, can be obtained using known methods in the art. (SeeMcCafferty et al., Nature 348:552-554 (1990); Clackson et al., Nature352:624-628; Marks et al., J. Mol. Biol. 222:581-597 (1991); Marks etal., Bio/Technology 10:779-783 (1992); Waterhouse et al., Nucleic. AcidsRes. 21:2265-2266 (1993); Morimoto et al., Journal of Biochemical andBiophysical Methods 24:107-117 (1992); Brennan et al., Science 229:81(1985); Carter et al., Bio/Technology 10:163-167 (1992); Kohler et al.,Nature 256:495 (1975); U.S. Pat. No. 4,816,567); Kilpatrick et al.,Hybridoma 16(4):381-389 (1997); Wring et al., J. Pharm. Biomed. Anal.19(5):695-707 (1999); Bynum et al., Hybridoma 18(5):407-411 (1999),Jakobovits et al., Proc. Natl. Acad. Sci. USA, 90:2551 (1993);Jakobovits et al., Nature, 362:255-258 (1993); Bruggemann et al., Yearin Immuno. 7:33 (1993); Barbas et al., Proc. Nat. Acad. Sci. USA91:3809-3813 (1994); Schier et al., Gene 169:147-155 (1995); Yelton etal., J. Immunol. 155:1994-2004 (1995); Jackson et. al., J. Immunol.154(7):3310-9 (1995); Hawkins et al., J. Mol. Biol. 226:889-896 (1992),U.S. Pat. Nos. 5,514,548, 5,545,806, 5,569,825, 5,591,669, 5,545,807; WO97/17852, all of which are herein incorporated by reference in theirentirety.)

Non-limiting examples of the antibody include, but not limited to,Muromonab-CD3 Abciximab, Rituximab, Daclizumab, Palivizumab, Infliximab,Trastuzumab, Etanercept, Basiliximab, Gemtuzumab ozogamicin,Alemtuzumab, Ibritumomab tiuxetan, Adalimumab, Alefacept, Omalizumab,Efalizumab, Tositumomob-I131, Cetuximab, Bevacizumab, Natalizumab,Ranibizumab, Panitumumab, Ecolizumab, Rilonacept, Certolizumab pegol,Romiplostim, AMG-531, CNTO-148, CNTO-1275, ABT-874, LEA-29Y, Belimumab,TACI-Ig, 2nd gen. anti-CD20, ACZ-885, Tocilizumab (Atlizumab),Mepolizumab, Pertuzumab, Humax CD20, CP-675, 206 (Ticilimumab), MDX-010,IDEC-114, Inotuzumab ozogamycin, HuMax EGFR, Aflibercept, VEGF Trap-Eye,HuMax-CD4, Ala-Ala, ChAglyCD3; TRX4, Catumaxomab, IGN101, MT-201,Pregovomab, CH-14.18, WX-G250, AMG-162, AAB-001, Motavizumab; MEDI-524,efumgumab, Aurograb®, Raxibacumab, 3rd gen. anti-CD20, LY2469298,Veltuzumab.

In some embodiments, when the protein is a monoclonal antibody, at leastone light chain of the monoclonal antibody, at least one heavy chain ofthe monoclonal antibody, or both may comprise an amino acid regionhaving an amino acid motif that can be recognized an isoprenoidtransferase.

In embodiments, the C-terminus of the light or heavy chain is modified.Also, the CH2 regions of the Fc region may be glycosylated.

In some embodiments, a C-terminus of a protein (a fragment, analog, orderivative thereof) can be attached to an amino acid motif that can berecognized by isoprenoid transferase. In other embodiments, theC-terminus can be modified. The modification can be (i) a deletion inthe carboxy terminus of the protein, (ii) an oligopeptide or polypeptideaddition in the carboxy terminus of the protein, or (iii) a deletion inthe carboxy terminus of the protein and an oligopeptide or polypeptideaddition in the carboxy terminus of the protein. In related embodiments,the modification can be attached to the amino acid motif.

The term “isoprenoid transferase” used herein refers to an enzyme thatcan recognize a certain amino acid motif at or near a C-terminus of aprotein and perform selective alkylation at thiol position(s) ofcysteine residue(s) of the certain amino acid motif by adding anisoprenoid unit(s) to the protein bearing the certain amino acid motif.

Examples of the isoprenoid transferase include farnesyltransferase(FTase) and geranylgeranyltransferase (GGTase), which involve thetransfer of a farnesyl or a geranyl-geranyl moiety to C-terminalcysteine(s) of the target protein, respectively. GGTase can beclassified into GGTase I and GGTase II. FTase and GGTase I can recognizea CAAX motif and GGTase II can recognize a XXCC, XCXC, or CXX motif, inwhich C represents cysteine, A represents an aliphatic amino acid, and Xrepresents an amino acid that determines the substrate specificity ofthe isoprenoid transferases (Nature Rev. Cancer 2005, 5(5), pp. 405-12;Nature Chemical Biology, 2010, 17, pp. 498-506; Lane K T, Bees L S,Structural Biology of Protein of Farnesyltransferase andGeranylgeranyltransferase Type I, Journal of Lipid Research, 47, pp.681-699 (2006); Patrick J. Kasey, Miguel C. Seabra; ProteinPrenyltransferases, The Journal of Biological Chemistry, Vol. 271, No.10, Issue of March 8, pp. 5289-5292 (1996), the contents of thesereferences are hereby incorporated by reference in their entirety).

In the present invention, isoprenoid transferases from a variety ofsources, e.g., humans, animals, plants, bacteria, virus, and the likecan be used. In some embodiments, naturally occurring isoprenoidtransferases can be used. In some other embodiments, naturally orartificially modified isoprenoid transferases can be used. For example,an isoprenoid transferase having at least one amino acid sequencenaturally changed (including post-translational modification), anaturally or artificially truncated form of a naturally occurringisoprenoid transferase, an isoprenoid transferase that has been modifiedby at least one of (His)-tag, GST, GFP, MBP, CBP, Isopeptag, BCCP,Myc-tag, Calmodulin-tag, FLAG-tag, HA-tag, Maltose binding protein-tag,Nus-tag, Glutathione-S-transferase-tag, Green fluorescent protein-tag,Thioredoxin-tag, S-tag, Softag 1, Softag 3, Strep-tag, SBP-tag, Ty-tag,and the like.

Isoprenoid transferases can recognize an isosubstrate as well as asubstrate. The isosubstrate refers to a substrate analog which has amodification in the substrate. Isoprenoid transferases alkylate acertain amino acid motif (e.g., CAAX motif) at a C-terminus of a protein(Benjamin P. Duckworth et al, ChemBioChem 2007, 8, 98; Uyen T. T. Nguyenet al, ChemBioChem 2007, 8, 408; Guillermo R. Labadie et al, J. Org.Chem. 2007, 72(24), 9291; James W. Wollack et al, ChemBioChem 2009, 10,2934, the contents of which are incorporated herein by reference.). Afunctionalized protein can be produced using an isoprenoid transferaseand an isosubstrate through alkylation at a C-terminal cysteine(s).

For example, the cysteine residue of a C-terminal CAAX motif can bereacted with an isosubstrate using an isoprenoid transferase. In certaincases, AAX can then be removed by a protease. The resulting cysteine canthen be methylated at the carboxy terminus by an enzyme. (Iran M. Bell,J. Med. Chem. 2004, 47(8), 1869, which is incorporated herein byreference.) In the case of some proteins, cysteinylation andglutathionylation through disulfide bond formation can occur due topost-translational modification. Such a disulfide bond, however, can bereduced when such alkylation occurs by isoprenoid transferases.

The proteins of the present invention can be made using any molecularbiology or cell biology method well known in the art. For example,transient transfection methods can be used. Genetic sequences encoding acertain amino acid motif that can be recognized by an isoprenoidtransferase can be inserted into a known plasmid vector using standardPCR technologies so as to express a protein (a fragment or analogthereof) having the certain amino acid motif at a C-terminus thereof. Assuch, a protein having at least one amino acid motif that can berecognized by an isoprenoid transferase can be expressed. The expressedprotein can then be enzymatically reacted with an isosubstrate of anisoprenoid transferase using the isoprenoid transferase to produce afunctionalized protein. The isosubstrate contains a functional group.

Once a protein having an amino acid motif that can be recognized by anisoprenoid transferase is expressed, it may be enzymatically reacted,using an isoprenoid transferase and at least one isosubstrate having afirst functional group (FG1), thereby producing a functionalizedprotein.

The term “functional group” used herein refers to a group that can leadto, e.g., 1,3-dipolar cycloaddition reactions, hetero-diels reactions,nucleophilic substitution reactions (e.g., of a ring opening reaction ofa heterocyclic electrophile such as epoxide, aziridine, cyclic sulfate,and aziridium), non-aldol type carbonyl reactions (e.g., formation ofoxime ethers, ureas, thioureas, aromatic heterocycles, hydrazones andamides), additions to carbon-carbon multiple bonds, oxidation reactions(e.g., epoxidation, aziridination, and sulfenyl halide addition), andclick chemistry. The functional group can include, but not limited to, afluorescent tag, a triazole, a maleimide, and a radioisotope (Angew.Chem. Int. Ed. 2001, 40, 2004-2021; Drug Discovery Today, 2003, 8(24),1128-1137; Chem. Rev. 2008, 108, 2952-3015, the contents of which areincorporated herein by reference.) In embodiments, the functional groupcan be an acetylene group and an azide group.

The functional group can be attached to a protein or an active agent viaat least one linker. In some embodiments, the linker is a linear linker.In some other embodiments, the linker is a branched linker. When thelink is a branched linker, active agents can be attached to all of thebranches. Each branch can have the same or different active agents. Insome embodiments, the linker can be cleavable. In some otherembodiments, it can be non-cleavable.

In some embodiments, a functionalized active agent is produced byattaching a second functional group (FG2) to an active agent. Exemplaryactive agents include, but are not limited to, a drug, a toxin, anaffinity ligand, a detection probe, or a combination thereof.

Exemplary drugs include, but are not limited to, erlotinib (TARCEVA;Genentech/OSI Pharm.), bortezomib (VELCADE; MilleniumPharm.),fulvestrant (FASLODEX; AstraZeneca), sutent (SUl11248; Pfizer),letrozole (FEMARA; Novartis), imatinib mesylate (GLEEVEC; Novartis),PTK787/ZK 222584 (Novartis), oxaliplatin (Eloxatin; Sanofi),5-fluorouracil (5-FU, leucovorin, rapamycin (Sirolimus, RAPAMUNE;Wyeth), lapatinib (TYKERB, GSK572016; GlaxoSmithKline), lonafarnib (SCH66336), sorafenib (BAY43-9006; Bayer Labs.), gefitinib (IRESSA;Astrazeneca), AG1478, AG1571 (SU 5271; Sugen), alkylating agents such asthiotepa and CYTOXAN® cyclophosphamide; alkyl sulfonates such asbusulfan, improsulfan and piposulfan; aziridines such as benzodopa,carboquone, meturedopa, and uredopa; ethylenimine and methylamelaminesincluding altretamine, triethylenemelamine, trietylenephosphoramide,triethiylenethiophosphoramide and trimethylolomelamine; acetogenins(especially, bullatacin and bullatacinone); camptothecin (including thesynthetic analogue topotecan); bryostatin; callystatin; CC-1065(including its adozelesin, carzelesin and bizelesin syntheticanalogues); cryptophycins (particularly cryptophycin 1 and cryptophycin8); dolastatin; duocarmycin (including the synthetic analogues, KW-2189and CB1-TM1); eleutherobin; pancratistatin; sarcodictyin; spongistatin;nitrogen mustards such as chlorambucil, chlornaphazine,cholophosphamide, estramustine, ifosfamide, mechlorethamine,mechlorethamine oxide hydrochloride, melphalan, novembichin,phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosoureasuch as carmustine, chlorozotocin, fotemustine, lomustine, nimustine,and ranimnustine; antibiotics such as the enediyne antibiotics (e.g,calicheamycin, especially calicheamycin gamma1 I and calicheamycinomegaI1 (see, e.g., Agnew, Chem Intl ed. Engl., 33: 183-186 (1994)) anddynemicin, including dynemicin A; bisphosphonate such as clodronate;esperamicin, neocarzinostatin chromophore and related chromoproteinenediyne antibiotic chromophores, aclacinomysins, actinomycin,antrmycin, azaserine, bleomycins, cactinomycin, carabicin, carninomycin,carzinophilin, chromomycins, dactinomycin, daunorubicin, detorubucin,6-diazo-5-oxo-L-norleucine, ADRLIMYCIN® doxorubicin (includingmorpholino-doxorubicin, cyanomorpholino-doxorubicin,2-pyrrolino-doxorubucin, liposomal doxorubicin and deoxydoxorubicin),epirubicin, esorubicin, marcellomycin, mitomycins such as mitomycin C,mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin,puromycin, quelamycin, rodorubicin, streptomigrin, streptozocin,tubercidin, ubenimex, zinostatin, and zorubicin; anti-metabolites suchas 5-fluorouracil(5-FU); folic acid analogues such as denopterin,methotrexate, pteropterin, trimetrexate; purine analogs such asfludarabine, 6-mercaptopurine, thiamiprine, and thiguanine; pyrimidineanalogs such as ancitabine, azacitidine, 6-azauridine, carmofur,cytarabine, dideoxyuridine, doxifluridine, enocitabine, and floxuridine;androgens such as calusterone, dromostanolone propionate, epitiostanol,mepitiostane, and testolactone; anti-adrenals such as aminoglutethimide,mitotane, and trilostane; folic acid replenisher such as folinic acid;aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil;amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine;diaziquone; elfornithine; elliptinium acetate; an epothilone; etoglucid;gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids suchas maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol;nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone;2-ethylhydrazide; procarbazine; PSK® polysaccharide complex (JHS NaturalProducts, Eugene, Oreg.); razoxane; rhizoxin; sizofiran; spirogermanium;tenuazonic acid; triaziquone; 2,2′,2″-trichlorotriethylamine;trichothecenes (especially T-2 toxin, verracurin A, roridin A andanguidine); urethane; vindesine; dacarbazine; mannomustine;mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside (‘Ara-C’);cyclophosphamide; thiotepa; taxoids, e.g., TAXOL® paclitaxel(Bristol-Myers Squibb Oncology, Princeton, N.J.) ABRAXANE™cremophor-free, albumin-engineered nanoparticle formulation ofpaclitaxel (American Pharmaceutical Partners, Schaumber, Ill.), andTAXOTERE® doxetaxel (Rhone-Poulenc Rorer, Antony, France); chloranbucil;gemcitabine; 6-thioguanine; mercaptopurine; platinum analogs such ascisplatin, carboplatin; vinblastine; platinum; etoposide, ifosfamide;mitoxantrone; vincristine; NAVELBINE® vinorelbine; novantrone;teniposide; edatrexate; daunomycin; aminopterin; xeloda; ibandronate;CPT-11; topoisomerase inhibitor RFS 2000; difluorometlhylornithine(DFMO); retinoids such as retinoic acid; capecitabine; andpharmaceutically acceptable salts, solvates, acids, or derivativesthereof.

Additional drugs include, but are not limited to, (i) anti-hormonalagents that act to regulate or inhibit hormone action on tumors such asanti-estrogens and selective estrogen receptor modulators (SERMs),including, for example, tamoxifen (including NOLVADEX® tamoxifen),raloxifene, droloxifene, 4-hydroxytamoxifen, trioxifene, keoxifene,LY117018, onapristone, and FAREATON® toremifene; (ii) aromataseinhibitors that inhibit the enzyme aromatase, which regulates estrogenproduction in the adrenal glands, such as, for example, 4(5)-imidazoles,aminoglutethimide, MEGASE® megestrol acetate, AROMASIN® exemestane,FEMARA® letrozole, and ARIMIDEX® anastrozole; (iii) anti-androgens suchas flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; aswell as troxacitabine (a 1,3-dioxolane nucleoside cytosine analog); (iv)aromatase inhibitors; (v) protein kinase inhibitors; (vi) lipid kinaseinhibitors; (vii) antisense oligonucleotides, particularly those thatinhibit expression of genes in signaling pathways implicated in aberrantcell proliferation, such as, for example, PKC-alpha, Raf, H-Ras; (viii)ribozyme, for example, VEGF inhibitor such as ANGIOZYME ribozyme andHER2 expression inhibitors; (ix) vaccines such as gene therapy vaccine;ALLOVECTIN® vaccine, LEUVECTIN vaccine and VAXID vaccine;PROLEUKIN®rIL-2; LURTOTECAN® topoisomerase 1 inhibitor; ABARELIX® rmRH;(x) an anti-angiogenic agent such as Bevacizumab (AVASTIN, Genentech);and (xi) pharmaceutically acceptable salts, solvates, acids, orderivatives thereof.

In some embodiments, cytokines can be used as the drug. Cytokines aresmall cell-signaling protein molecules that are secreted by numerouscells and are a category of signaling molecules used extensively inintercellular communication. They include monokines, lymphokines,traditional polypeptide hormones, and the like. Examples of cytokinesinclude, but are not limited to, growth hormone such as human growthhormone, N-methionyl human growth hormone, and bovine growth hormone;parathyroid hormone; thyroxine; insulin; proinsulin; relaxin;prorelaxin; glycoprotein hormones such as follicle stimulating hormone(FSH), thyroid stimulating hormone (TSH), and luteinizing hormone (LH);hepatic growth factor fibroblast growth factor; prolactin; placentallactogen; tumor necrosis factor-α and -β; mullerian-inhibitingsubstance; mouse gonadotropin-associated peptide; inhibin; activin;vascular endothelial growth factor; integrin; thrombopoietin (TPO);nerve growth factors such as NGF-β; platelet-growth factor; transforminggrowth factors (TGFs) such TGF-α and TGF-β; insulin-like growth factor-Iand -II; erythropoietin (EPO); osteoinductive factors; interferons suchas interferon-α, -β, and -γ; colony stimulating factors (CSFs) such asmacrophage-CSF (M-CSF); granulocyte-macrophage-CSF (GM-CSF); andgranulocyte-CSF (G-CSF); interleukins (ILs) such as IL-1, IL-1α, IL-2,IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12; a tumornecrosis factor such as TNF-α and TNF-β; and other polypeptide factorsincluding LIF and kit ligand (KL). As used herein, the term cytokinealso includes proteins from natural sources or from recombinant cellculture and biologically active equivalents of the native sequencecytokines.

The term “toxin” refers to a poisonous substance produced within livingcells or organisms. Toxins can be small molecules, peptides or proteinsthat are capable of causing disease on contact with or absorption bybody tissue interacting with biological macromolecules such as enzyme orcellular receptors. Toxins include plant toxins and animal toxins.Examples of animal toxins include, but are not limited to, diphtheriatoxin, botulinum toxin, tetanus toxin, dysentery toxin, cholera toxin,tetrodotoxin, brevetoxin, ciguatoxin. Examples of plant toxins include,but are not limited to, ricin and AM-toxin.

Examples of small molecule toxins include, but are not limited to,auristatin, geldanamycin (Kerr et al., 1997, Bioconjugate Chem.8(6):781-784), maytansinoids (EP 1391213, ACR 2008, 41, 98-107),calicheamycin (US 2009105461, Cancer Res. 1993, 53, 3336-3342),daunomycin, doxorubicin, methotrexate, vindesine, SG2285 (Cancer Res.2010, 70(17), 6849-6858), dolastatin, dolastatin analogue's auristatin(US563548603), cryptophycin, camptothecin, rhizoxin derivatives, CC-1065analogues or derivatives, duocarmycin, enediyne antibiotics,esperamicin, epothilone, and toxoids. Toxins can exhibit cytotoxicityand cell growth-inhibiting activity by tubulin binding, DNA binding,topoisomerase suppression, and the like.

The term “ligand” refers to a molecule that can form a complex with atarget biomolecule. An example of a ligand is a molecule that isattached to a predetermined position of a targeted protein and transmitsa signal. It can be a substrate, an inhibitor, a stimulating agent, aneurotransmitter, or a radioisotope.

“Detectable moiety” or a “label” refers to a composition detectable byspectroscopic, photochemical, biochemical, immunochemical, radioactive,or chemical means. For example, useful labels include ³²P, ³⁵S,fluorescent dyes, electron-dense reagents, enzymes (e.g., as commonlyused in an ELISA), biotin-streptavidin, dioxigenin, haptens and proteinsfor which antisera or monoclonal antibodies are available, or nucleicacid molecules with a sequence complementary to a target. The detectablemoiety often generates a measurable signal, such as a radioactive,chromogenic, or fluorescent signal, that can be used to quantify theamount of bound detectable moiety in a sample. Quantitation of thesignal is achieved by, e.g., scintillation counting, densitometry, flowcytometry, ELISA, or direct anlaysis by mass spectreometry of intact orsubsequently digested peptides (one or more peptide can be assessed).Persons of skill in the art are familiar with techniques for labelingcompounds of interest, and means for detection. Such techniques andmethods are conventional and well-known in the art.

The term “probe” as used herein refers to a material that can (i)provide a detectable signal, (ii) can interact a first probe or a secondprobe to modify a detectable signal provided by the first or secondprobe, such as fluorescence resonance energy transfer (FRET), (iii)stabilize the interaction with an antigen or a ligand or increase thebinding affinity; (iv) affect electrophoresis mobility or cell-intrudingactivity by a physical parameter such as charge, hydrophobicity, etc.,or (v) control ligand affinity, antigen-antibody binding, or ioniccomplex formation.

Once the functionalized protein and the functionalized active agent areproduced, they are reacted with each other, thereby producing theprotein-active agent conjugate. In embodiments, the reaction between thefunctionalized protein and the functionalized active agent may be aclick chemistry reaction or via a hydrazone and/or oxime formation. Inembodiments, the FG1 is an azide group and the FG2 is an acetylenegroup, or vice versa. In other embodiments, the FG1 may be an aldehydeor ketone group and the FG2 is a hydrazine or hydroxylamine, or viceversa.

Click chemistry reactions are conducted in a mild condition, making itpossible to handle proteins easily. Click chemistry reaction shows veryhigh reaction specificity. Thus, even if a protein has other functionalgroups (e.g., side chain residue or at a C-terminus or N-terminus),these functional groups are not affected by the click chemistryreaction. For example, a click chemistry reaction between an acetylenegroup and an azide group of a protein can occur while other functionalgroups of the protein are not affected by the click chemistry reaction.In addition, a click chemistry reaction can specifically occur withoutbeing affected by the kind of ligand involved. In some cases, the ligandcan be selected to improve overall reaction efficiency. For example,azide-acetylene click chemistry can produce a triazole at a high yield(Rhiannon K. Iha et al, Chem. Rev. 2009, 109, 5620; Morten Meldal andChristian Wenzel Tornoe, Chem Rev., 2008, 108, 2952; Hartmuth C. Kolb etal, Angew. Chemie Int. Ed. Engl., 2001, 40, 2004, all of which areincorporated herein by reference.)

Azide and acetylene groups are functional groups that do not exist inamino acid sequences of naturally occurring proteins. If a conjugationreaction occurs using these functional groups, none of the side chainresidues and none of the N-terminal or C-terminal functional groups areaffected by the click chemistry reaction. Accordingly, a protein-activeagent conjugate in which an active agent is conjugated at a targetedposition(s) can be produced.

When the protein is an antibody, all or a part of the antibody can bereduced to a single chain during alkylation by an isoprenoidtransferase. The single chain can be oxidized to form a H₂L₂-formantibody due to an oxidizer used in the click chemistry reaction.

As the antibody has 4 chains (2H+2L), alkylation can be made at 1-4positions per antibody. The number of the active agents can be more than4 since a plurality of the active agents can be attached to a linker.

In certain embodiments, when the amino acid motif that can be recognizedby the isoprenoid transferase is CAAX, the method may further includeremoving AAX. In other embodiments, the method may further includeadding a methyl group at the C-terminus after removing AAX (Journal ofLipid Research, 2006, 47, 681-699, which is incorporated herein byreference.).

Embodiment 2

A method for preparing a protein-active agent conjugate according toanother embodiment comprises: (a) expressing a protein having an aminoacid motif that can be recognized by an isoprenoid transferase; (b)attaching an isosubstrate of an isoprenoid transferase to an activeagent; and (c) enzymatically reacting, using the isoprenoid transferase,the expressed protein with the active agent attached to theisosubstrate.

In this embodiment, once a protein having an amino acid motif that canbe recognized by an isoprenoid transferase is expressed, the protein isreacted with an active agent attached to an isosubstrate of theisoprenoid transferase. In this case, thiol-maleimide conjugation mayoccur. However, even if thiol-maleimide conjugation occurs, the activeagents are conjugated at the targeted positions only according to thepresent invention. Accordingly, a problem associated with the prior artthat a non-homogeneous mixture is produced is avoided.

2. Protein-Active Agent Conjugates

In another aspect, the present invention provides a protein-active agentconjugate comprising a protein having an amino acid motif that can berecognized by an isoprenoid transferase, wherein the active agent iscovalently linked to the protein at the amino acid motif.

One of ordinary skill in the art is readily able to select a proteinthat selectively binds a target of interest (e.g., a target cell in asubject). Exemplary proteins include, but are not limited to antibodiesor fragments of an antigen-binding protein that specifically bind to thetarget of interest.

CAAX Protein (CAAX Antibody)

An example of a protein-active agent conjugate prepared by a method ofthe present invention is represented by the following formula (I), inwhich the protein is an antibody (fragment or analog thereof) (Ab), theactive agent is a drug (D), and the amino acid motif that can berecognized by an isoprenoid transferase is CAAX.

Ab(M) represents that the antibody or fragment thereof, which cancomprise a modification. The modification can be (i) a deletion in thecarboxy terminus of the antibody or fragment thereof, (ii) anoligopeptide or polypeptide addition in the carboxy terminus of theantibody or fragment thereof; and (iii) a deletion in the carboxyterminus of the antibody or fragment thereof and an oligopeptide orpolypeptide addition in the carboxy terminus of the antibody or fragmentthereof. Q represents a linker. The linker can be a linear linker or abranched linker. In an embodiment, the linker can include a firstfunctional group (FG1). n₁, n₂, and m can be appropriately determineddepending on the antibody, the amino acid motif, linker, active agent,etc. Preferably, n₁ and n₂ are independently an integer of 1 to 4 and mis an integer of 1 to 16.

In some embodiments, the linker can be represented by the followingformula (II):

P₁ and Y is independently a group containing a first functional group(FG1). The FG 1 can be selected from the group consisting of: acetylene,azide, aldehyde, hydroxylamine, hydrazine, ketone, nitrobenzofurazan(NBD), dansyl, fluorescein, biotin, and Rhodamine. L₁ is(CH₂)_(r)X_(q)(CH₂)_(p), in which X is oxygen, sulfur, —NR₁—, —C(O)NR₁—,—NR₁C(O)—, —NR₁SO₂—, —SO₂NR₁—, —(CH═CH)—, or acetylene; R₁ is hydrogen,C₁₋₆ alkyl, C₁₋₆ alkyl aryl, or C₁₋₆ alkyl heteroaryl; r and p isindependently an integer of 0 to 6; q is an integer of 0 to 1; and n isan integer of 1 to 4.

In some certain embodiments, the drug (D) can be attached to the linkervia a group containing a second functional group (FG2) that can reactwith the FG1. The FG2 can be selected from the group consisting of:acetylene, hydroxylamine, azide, aldehyde, hydrazine, ketone, and amine.

In some certain embodiments, the drug (D) can be attached to the groupcontaining an FG2 via —(CH₂)_(r)X_(q)(CH₂)_(p)— or—[ZCH₂CH₂O(CH₂CH₂O)_(w)CH₂CH₂Z]—, in which X is oxygen, sulfur, —NR₁—,—C(O)NR₁—, —NR₁C(O)—, —NR₁SO₂—, or —SO₂NR₁—; Z is oxygen, sulfur or NR₁;R₁ is hydrogen, C₁₋₆ alkyl, C₁₋₆ alkyl aryl, or C₁₋₆ alkyl heteroaryl; rand p is independently an integer of 0 to 6; q is an integer of 0 to 1;and w is an integer of 0 to 6.

In some certain embodiments, (i) a peptide(s) that can be cleaved bycathepsin B or (ii) a glucuronide that can be cleaved by β-glucuronidasecan be attached to the —(CH₂)_(r)X_(q)(CH₂)_(p)— or—[ZCH₂CH₂O(CH₂CH₂O)_(w)CH₂CH₂Z]—.

In some certain embodiments, a non self-immolative group or aself-immolative group can be attached to the (i) peptide(s) that can becleaved by cathepsin B or (ii) glucuronide that can be cleaved byβ-glucuronidase. Non-limiting examples of the self-immolative group maybe aminophenylmethyloxycarbonyl and hydroxyphenylmethyloxycarbonyl.

In some certain embodiments, the peptide that can be cleaved bycathepsin B is represented by the following formula (III):

In some certain embodiments, the glucuronide that can be cleaved byβ-glucuronidase is represented by the following formula (IV):

3. Compositions

In still another aspect, the present invention provides compositionscomprising a protein-active agent conjugate described herein. Inembodiments, the compositions are used for delivering an active agent toa target cell in a subject. In embodiments, the compositions are used totreat a subject in need thereof (i.e., in need of the active agent).

The preparation of such compositions is known to one skilled in the art,and such compositions can be delivered in vivo to a subject.

In aspects, the compositions are prepared in an injectable form, eitheras a liquid solution or as a suspension. Solid forms suitable forinjection may also be prepared as emulsions, or with the polypeptidesencapsulated in liposomes. The protein-active agent conjugates can becombined with a pharmaceutically acceptable carrier, which includes anycarrier that does not induce the production of antibodies harmful to thesubject receiving the carrier. Suitable carriers typically compriselarge macromolecules that are slowly metabolized, such as proteins,polysaccharides, polylactic acids, polyglycolic acids, polymeric aminoacids, amino acid copolymers, lipid aggregates, and the like. Suchcarriers are well known to those skilled in the art.

The compositions of the invention can also contain diluents, such aswater, saline, glycerol, and ethanol. Auxiliary substances may also bepresent, such as wetting or emulsifying agents, pH buffering substances,and the like. Proteins may be formulated into the vaccine as neutral orsalt forms. The compositions can be administered parenterally, byinjection; such injection may be either subcutaneously orintramuscularly. Additional formulations are suitable for other forms ofadministration, such as by suppository or orally. Oral compositions maybe administered as a solution, suspension, tablet, pill, capsule, orsustained release formulation.

The compositions are administered in a manner compatible with the doseformulation. The composition comprises a therapeutically effectiveamount of the protein-active agent conjugate. By a therapeuticallyeffective amount is meant a single dose, or a composition administeredin a multiple dose schedule, that is effective for the treatment orprevention of a disease or disorder. The dose administered will vary,depending on the subject to be treated, the subject's health andphysical condition, the degree of protection desired, and other relevantfactors. Precise amounts of the active ingredient required will dependon the judgment of the practitioner.

4. Methods of Using Protein-Active Agent Conjugates and Compositions

In a further aspect, the present invention provides a method fordelivering an active agent to a target cell in a subject, the methodcomprising administering the protein-active agent conjugate or thecomposition. In a still further aspect, the present invention provides amethod of treating a subject in need thereof (i.e., a subject in need ofthe active agent), the method comprising administering an effectiveamount of the protein-active agent conjugate or a composition comprisingthe conjugate to the subject.

In embodiments, a protein-active agent conjugate (e.g., antibody-drugconjugate) or a composition comprising the conjugate in atherapeutically effective amount can be administered to a patientsuffering from a cancer or tumor to treat the cancer or tumor.

In embodiments, a protein-active agent conjugate (e.g., antibody-drugconjugate) or a composition comprising the conjugate in atherapeutically effective amount can be administered to a patient totreating or preventing an infection by a pathogenic agent (e.g., avirus, a bacteria, a fungus, a parasite, and the like). Such methodsinclude the step of administering to the mammal a therapeutic orprophylactic amount of an amount of the conjugate sufficient to treatthe disease or disorder or symptom thereof, under conditions such thatthe disease or disorder is prevented or treated.

In some embodiments, the protein-active agent conjugate or compositioncan be administered in the form of a pharmaceutically acceptable salt orsolvate thereof. In some embodiments, it can be administered with apharmaceutically acceptable carrier, a pharmaceutically acceptableexcipient, and/or a pharmaceutically acceptable additive. Thepharmaceutically effective amount and the type of the pharmaceuticallyacceptable salt or solvate, excipient and additive can be determinedusing standard methods (Remington's Pharmaceutical Sciences, MackPublishing Co., Easton, Pa., 18^(th) edition, 1990).

The term “therapeutically effective amount” with regard to a cancer ortumor means an amount that can decrease the number of cancer cells;decrease the size of cancer cells; prohibit cancer cells from intrudingperipheral systems or decrease the intrusion; prohibit cancer cells frombeing spreading to other systems or decrease the spreading; prohibitcancer cells from growing; and/or ameliorate at least one symptomsrelated to the cancer. In the treatment of a cancer, the effectivenessof a drug can be assessed by time to tumor progression (TTP) and/orresponse rate (RR).

The term “therapeutically effective amount” with regard to infection bya pathogenic agent means an amount that can prevent, treat, or reducethe symptoms associated with infection.

The term “pharmaceutically acceptable salts” used herein includesorganic salts and inorganic salts. Examples thereof include, but are notlimited to, hydrochloride, hydrobromide, hydroiodide, sulfate, citrate,acetate, oxalate, chloride, bromide, iodide, nitrate, bisulfate,phosphate, acidic phosphate, isonicotinate, lactate, salicylate, acidiccitrate, tartrate, oleate, tannate, pantonate, bitartrate, ascorbate,succinate, maleate, gentisinate, fumarate, gluconate, glucoronate,saccharate, formate, benzoate, glutamate, methane sulfonate, ethanesulfonate, benzene sulfonate, p-toluene sulfonate, and pamoate (i.e.,1,1′-methylenebis-(2-hydroxy-3-naphthoate)). A pharmaceuticallyacceptable salt can include another molecule (e.g., acetate ions,succinate ions, and other counter ions, etc.). It also can include atleast one charged atom. It also can include at least one counter ion.

Exemplary solvates that can be used to pharmaceutical acceptablesolvates of the compounds according to the present invention include,but not limited to, water, isopropanol, ethanol, methanol, DMSO, ethylacetate, acetic acid, and ethanol amine.

EXAMPLES

The following examples illustrate the invention and are not intended tolimit the same.

Example 1: Preparation of Ab(M)-CAAX

1-1. Construction, Expression, and Purification of Herceptin-CAAX

Modified Herceptin antibodies were generated using standard recombinantDNA technology and PCR cloning protocols with pNATABH::Herceptin HCplasmid or pNATABL::Herceptin LC plasmid. Recombinant plasmids wereexpressed in an HEK293E cell line by transient transfection. Theantibodies were separated and purified by protein A columnchromatography.

Construction of Herceptin-HC-GCVIM (“Herceptin-HC-GCVIM” disclosed asSEQ ID NO: 8) and Herceptin-LC-GCVIM (“Herceptin-LC-GCVIM” disclosed asSEQ ID NO: 11)

Modified Herceptin antibodies were generated using standard PCR cloningprotocols. Generally, Herceptin-HC-GCVIM (“Herceptin-HC-GCVIM” disclosedas SEQ ID NO: 8) and Herceptin-LC-GCVIM (“Herceptin-LC-GCVIM” disclosedas SEQ ID NO: 11) plasmids were constructed by inserting a DNA sequenceencoding a CAAX motif (e.g., GCVIM (SEQ ID NO: 1), G₅CVIM (SEQ ID NO:2), G₇CVIM (SEQ ID NO: 3), G₁₀CVIM (SEQ ID NO: 4), or G₁₀CVLL (SEQ IDNO: 6)), to the C-terminus of the heavy chain or light chain encoded inthe pNATABH::Herceptin HC or pNATABH::Herceptin LC plasmid.

For example, a SacII recognition sequence is present at amino acid 172in the C-terminus of the human IgG1-Fc region. Accordingly, a forwardprimer was designed to bind the SacII site in the Fc region. The DNAsequence to be inserted (e.g., the 15-mer encoding GCVIM (SEQ ID NO: 1)5-mer sequence) was added to a reverse primer specific for theFc-C-terminal end. The forward and reverse primers were used to amplifya PCR product, and the resultant product was purified using a PCRpurification kit. As the reverse primer contained an XhoI site, the PCRproduct was digested with SacII and XhoI. Likewise, thepNATABH::Herceptin HC plasmid was digested with SacII and XhoI. Thedigested backbone was purified using a gel purification kit and ligatedwith the digested PCR product. Ligation was performed by appropriatelyadjusting the ratio of the vector and the insert, and the ligationproduct was transformed into competent bacterial cells for screening.Herceptin-HC-GCVIM (“Herceptin-HC-GCVIM” disclosed as SEQ ID NO: 8) andHerceptin-LC-GCVIM (“Herceptin-LC-GCVIM” disclosed as SEQ ID NO: 11)plasmids were prepared from sequenced clones.

The amino acid sequences from the resultant plasmids are shown in FIGS.1-10. Sections 1-4 and 1-7 below provide a detailed description of eachof the constructs.

Expression and purification of Herceptin-HC-GCVIM (“Herceptin-HC-GCVIM”disclosed as SEQ ID NO: 8) and Herceptin-LC-GCVIM (“Herceptin-LC-GCVIM”disclosed as SEQ ID NO: 11)

HEK293E cells were cultured in DMEM/10% FBS media on 150 mm plates(#430599, Corning USA) until 70-80% confluency. 13 μg of DNA and 26 μgof PEI (#23966, Polysciences, USA) were mixed in a ratio of 1:2,incubated at RT for about 20 minutes, and then added to the HEK293Ecells. After 16-20 hours, the media was replaced with serum free media(No FBS DMEM (#SH30243.01, Hyclone Thermo., USA)) and supernatant wascollected every two or three days.

The supernatants were filtered with a 0.22 um top-filter (#PR02890,Millipore, USA) and then bound to 500 μl of protein A bead (#17-1279-03,GE healthcare Sweden) packed in a 5 mL column. Using a peristaltic pump,overnight binding was performed at 0.9 mL/min at 4° C. The column waswashed with 100 mL or greater of PBS (#70011, Gibco, USA). Bound proteinwas then eluted with 0.1M Glycine-HCl (#G7126, Sigma, USA) into 6fractions and neutralized with 1M Tris (#T-1503, Sigma, USA)(pH 9.0).The protein was quantified. 2 or 3 fractions containing the protein werecollected and concentrated with Amicon Ultra filter units (#UFC805024,Millipore, USA). Buffer was changed about 10 times with 1×PBS (#70011,Gibco, USA). The protein product was confirmed to be Herceptin-HC-GCVIM(“Herceptin-HC-GCVIM” disclosed as SEQ ID NO: 8) or Herceptin)-LC-GCVIM(“Herceptin-LC-GCVIM” disclosed as SEQ ID NO: 11) by Western blot. Toidentify a protein band containing Herceptin, ImmunoPure peroxidaseconjugated goat anti-human IgG Fc (#31413, Pierce, USA) was used. Uponpurification, 1-2 mg of Herceptin-HC-CGVIM (“Herceptin-HC-GCVIM”disclosed as SEQ ID NO: 8) or Herceptin-LC-GCVIM (“Herceptin-LC-GCVIM”disclosed as SEQ ID NO: 11) was obtained from 1 L of cell culturemedium.

The Herceptin-HC-GCVIM (“Herceptin-HC-GCVIM” disclosed as SEQ ID NO: 8)and Herceptin-LC-GCVIM (“Herceptin-LC-GCVIM” disclosed as SEQ ID NO: 11)products were also analyzed with an Agilent bioanalyzer. Briefly, 8 μlof purified protein sample (approx. 1 mg/ml) was analyzed using theAgilent Protein 230 Kit (5067-1515 Agilent Technologies, USA). Theprotein sample was separated into 2 fractions (4 μl each). 2 μl ofnon-reducing buffer or reducing buffer was added to each sample. Thesample was heated at 95-100° C. for 5 minutes and cooled with ice to 4°C. After spin-down, 84 μl of deionized water was added to the sample andladder and vortexed. Thereafter, the sample was loaded and analyzed withthe kit per manufacturer's instructions.

1-2. Construction, Expression and Purification of Anti cMET-CAAX

Modified anti cMET-CAAX antibodies were also prepared by theabove-described methods. For example, modified anti cMET-CAAX antibodieswere generated using standard recombinant DNA technology and PCR cloningprotocols with pPMC-C1A5 plasmid. Recombinant plasmids were expressed inan HEK293T cell line by transient transfection. The antibodies wereseparated and purified by protein A column chromatography.

1-3. Herceptin-HC-G_(n)CVIM (“G_(n)CVIM” Disclosed as SEQ ID NO: 5)

Herceptin-HC-GCVIM (“Herceptin-HC-GCVIM” disclosed as SEQ ID NO: 8),Herceptin-HC-G₅CVIM (“Herceptin-HC-G₅CVIM” disclosed as SEQ ID NO: 12),Herceptin-HC-G₇CVIM (“Herceptin-HC-G₇CVIM” disclosed as SEQ ID NO: 16),and Herceptin-HC-G₁₀CVIM (“Herceptin-HC-G₁₀CVIM” disclosed as SEQ ID NO:20) antibodies were prepared. The antibodies, respectively, have a5-mer(GCVIM) (SEQ ID NO: 1), a 9-mer(G₅CVIM) (SEQ ID NO: 2), an11-mer(G₇CVIM) (SEQ ID NO: 3), or a 14-mer(G₁₀CVIM) (SEQ ID NO: 4)sequence at the C-terminus of the heavy chain (FIGS. 1, 3, 5, and 7).

1-4. Herceptin-LC-G_(n)CVIM (“G_(n)CVIM” Disclosed as SEQ ID NO: 5)

Herceptin-LC-GCVIM (“Herceptin-LC-GCVIM” disclosed as SEQ ID NO: 11),Herceptin-LC-G₅CVIM (“Herceptin-LC-G₅CVIM” disclosed as SEQ ID NO: 15),Herceptin-LC-G₇CVIM (“Herceptin-LC-G₇CVIM” disclosed as SEQ ID NO: 19),and Herceptin-LC-G₁₀CVIM (“Herceptin-LC-G₁₀CVIM” disclosed as SEQ ID NO:23) antibodies were prepared. The antibodies, respectively, have a 5-mer(GCVIM) (SEQ ID NO: 1), a 9-mer(G₅CVIM) (SEQ ID NO: 2), an11-mer(G₇CVIM) (SEQ ID NO: 3), or a 14-mer(G₁₀CVIM) (SEQ ID NO: 4)sequence at the C-terminus of the light chain (FIGS. 2, 4, 6, and 8).

1-5. Herceptin-HC-G₁₀CVLL (“Herceptin-HC-G₁₀CVLL” Disclosed as SEQ IDNO: 24)

A Herceptin-C-G₁₀CVLL (“Herceptin-HC-G₁₀CVLL” disclosed as SEQ ID NO:24) antibody was prepared. The antibody has a 14-mer(G₁₀CVLL) (SEQ IDNO: 6) sequence at the C-terminus of the heavy chain (FIG. 9).

1-6. Herceptin-LC-G₁₀CVLL (“Herceptin-LC-G₁₀CVLL” Disclosed as SEQ IDNO: 27)

A Herceptin-LC-G₁₀CVLL (“Herceptin-LC-G₁₀CVLL” disclosed as SEQ ID NO:27) antibody was prepared. The antibody has a 14-mer(G₁₀CVLL) (SEQ IDNO: 6) sequence at the C-terminus of the light chain (FIG. 10).

1-7. Anti cMET-HC-G_(n)CVIM (“G_(n)CVIM” Disclosed as SEQ ID NO: 5)

Anti cMET-HC-G₇CVIM (“G₇CVIM” disclosed as SEQ ID NO: 3) and anticMET-HC-G₁₀CVIM (“G₁₀CVIM” disclosed as SEQ ID NO: 4) antibodies wereprepared. The antibodies, respectively, have an 11-mer(G₇CVIM) (SEQ IDNO: 3), or a 14-mer(G₁₀CVIM) (SEQ ID NO: 4) sequence at the C-terminusof the heavy chain (not shown). FIG. 11 shows an SDS-PAGE gel analyzingthe anti cMET-HC-G₇CVIM (“G₇CVIM” disclosed as SEQ ID NO: 3) and anticMET-HC-G₁₀CVIM (“G₁₀CVIM” disclosed as SEQ ID NO: 4) antibodies.

1-8. Anti cMET-LC-G_(n)CVIM (“G_(n)CVIM” Disclosed as SEQ ID NO: 5)

Anti cMET-LC-G₇CVIM (“G₇CVIM” disclosed as SEQ ID NO: 3) and anticMET-LC-G₁₀CVIM (“G₁₀CVIM” disclosed as SEQ ID NO: 4) antibodies wereprepared. The antibodies, respectively, have an 11-mer(G₇CVIM) (SEQ IDNO: 3), or a 14-mer(G₁₀CVIM) (SEQ ID NO: 4) sequence at the C-terminusof the light chain (not shown). FIG. 11 shows an SDS-PAGE gel analyzingthe anti cMET-LC-G₇CVIM (“G₇CVIM” disclosed as SEQ ID NO: 3) and anticMET-LC-G₁₀CVIM (“G₁₀CVIM” disclosed as SEQ ID NO: 4) antibodies.

Example 2: Functionalization of AB(M)-CAAX

2-1. Geranyl Alkyne Diphosphate (B, LCB14-0501)

The above-referenced compound was prepared in 6 steps with geraniol as astarting material by a method similar to the method described inChembioChem 207, 8, 98-105, the contents of which are herebyincorporated by reference in their entirety.

(B) ¹H NMR (600 MHz, D₂O) δ 5.38 (t, J=7.8 Hz, 1H), 5.30 (t, J=7.8 Hz,1H), 4.31 (brs, 2H), 3.96 (m, 2H), 3.84 (s, 2H), 2.70 (bs, 1H), 2.07 (m,2H), 1.98 (m, 2H), 1.56 (s, 3H), 1.48 (s, 3H)

2-2. Decadienyl Propargyl Ether Diphosphate (F, LCB14-0511) andDecadienyl Azide Diphosphate (G, LCB14-0512)

Acetoxydecadienyl aldehyde (C) was prepared from farnesol in 5 steps.From the compound (C), the compounds (D) and (E) were prepared in 6steps and 5 steps, respectively. From the compounds (D) and (E), theabove-referenced compounds (F) and (G) were prepared by a method similarto the method described in the section 2-1 above. The compounds (C),(D), and (E) were prepared by a method similar to the method describedin JOC 2007, 72(24), 9291-9297, the contents of which are herebyincorporated by reference in their entirety.

(F): ¹H NMR (600 MHz, D₂O) δ 5.44 (t, J=6 Hz, 1H), 5.22 (t, J=6 Hz, 1H),4.46 (t, J=8.4 Hz, 2H), 4.16 (t, J=2.4 Hz, 2H), 3.55 (m, 2H), 2.85 (m,1H), 2.15 (m, 2H), 2.09 (t, J=7.2 Hz, 2H), 2.03 (t, J=7.2 Hz, 2H),1.70˜1.65 (m, 5H), 1.60 (s, 3H)

(G): ¹H NMR (600 MHz, D₂O) δ 5.43 (t, J=6.6 Hz, 1H), 5.23 (t, J=6.6 Hz,1H), 4.40 (t, J=6 Hz, 2H), 3.26 (t, J=6.0 Hz, 2H), 2.15 (m, 2H),2.10˜2.04 (m, 4H), 1.70˜1.65 (m, 5H), 1.60 (s, 3H)

2-3. NBD-GPP

Tris-ammonium[3,7-dimethyl-8-(7-nitro-benzo[1,2,5]oxadiazol-4-ylamino)-octa-2,6-diene-1]pyrophosphate(NBD-GPP) was prepared by a method similar to the method described inJACS 2006, 128, 2822-2835, the contents of which are hereby incorporatedby reference in their entirety.

¹H NMR (600 MHz, D₂O) δ 8.51 (d, J=9 Hz, 1H), 6.37 (d, J=9 Hz, 1H), 5.50(t, J=6.6 Hz, 1H), 5.42 (t, J=6.6 Hz, 1H), 4.43 (t, J=6.6 Hz, 2H), 4.08(s, 2H), 2.22 (m, 2H), 2.10 (t, J=7.2 Hz, 2H), 1.69 (s, 6H)

2-4. Glucuronide Linker-MMAF (LCB14-0592)

Compound 2

To a solution of D-glucurono-6,3-lactone (19 g, 107.88 mmol) in methanol(250 mL) under nitrogen atmosphere was slowly added a solution of NaOH(100 mg) in methanol (100 mL). The resulting mixture was stirred for 2hours. A solution of NaOH (200 mg) in methanol (15 mL) was added. Theresultant was stirred for 3 hours. Methanol was removed under reducedpressure. At 10° C. or lower, pyridine (50 mL) and acetic anhydride(Ac₂O, 54 mL) were sequentially added. The resulting mixture was stirredat room temperature for 4 hours. After the reaction was completed, theresulting mixture was concentrated under reduced pressure, and subjectedto column chromatography to give the compound 2 (20 g, 50%) as a solid.

¹H NMR (600 MHz, CDCl₃) δ 5.77 (d, J=7.8 Hz, 1H), 5.31 (t, J=9.6 Hz,1H), 5.24 (t, J=9.6 Hz, 1H), 5.14 (m, 1H), 4.17 (d, J=9 Hz, 1H), 3.74(s, 3H), 2.12 (s, 3H), 2.04 (m, 9H)

Compound 3

The compound 2 (5 g, 13.28 mmol) was added to a solution of 33% HBr inAcOH (20 mL) at 0° C. The resulting mixture was stirred for 2 hours atroom temperature. After the reaction was completed, the resultingmixture was diluted by toluene (50 mL). The resulting mixture wasconcentrated under reduced pressure. Ethyl acetate (100 mL) andsaturated NaHCO₃ solution (100 mL) were added to extract an organiclayer. The thus-obtained organic layer was dried with anhydrous sodiumsulfate to give the compound 3 (5.27 g, 100%).

¹H NMR (600 MHz, CDCl₃) δ 6.64 (d, J=3.6 Hz, 1H), 5.61 (t, J=3.6 Hz,1H), 5.24 (t, J=3.6 Hz, 1H), 4.85 (m, 1H), 4.58 (d, J=10.2 Hz, 1H), 3.76(s, 3H), 2.10 (s, 3H), 2.06 (s, 3H), 2.05 (s, 3H)

Compound 4

A solution of the compound 3 (4 g, 10.07 mmol) and2,4-dihydroxybenzaldehyde (1.67 g, 12.084 mmol) in acetonitrile (30 mL)was treated sequentially with molecular sieve (5 g) and Ag₂O (9.33 g,40.28 mmol). The resulting mixture was stirred for 3 hours at roomtemperature. After the reaction was completed, the solid was filteredoff and the filtrate was concentrated under reduced pressure. Theresidue was subjected to column chromatography to give the compound 4 (2g, 43.5%).

¹H NMR (400 MHz, CDCl₃) δ 11.38 (s, 1H), 9.77 (s, 1H), 7.48 (d, J=8.4Hz, 1H), 6.61 (dd, J=8.4, 2.0 Hz, 1H), 6.53 (d, J=2.0 Hz, 1H), 5.36˜5.25(m, 4H), 4.23 (m, 1H), 3.73 (s, 1H), 2.06 (s, 9H)

Compound 5

A solution of the compound 4 (1 g, 2.20 mmol) in acetone (10 mL) wastreated with potassium carbonate (760 mg, 5.50 mmol) and 80% propargylbromide in toluene (735 μL, 6.60 mmol). The resulting mixture wasstirred at 45° C. for 12 hours. After the reaction was completed, ethylacetate (100 mL) and distilled water (100 mL) were added. Thethus-obtained organic layer was dried with anhydrous sodium sulfate andconcentrated under reduced pressure.

The residue was subjected to column chromatography to give the compound5 (930 mg, 87%).

¹H NMR (600 MHz, CDCl₃) δ 10.33 (s, 1H), 7.83 (d, J=9 Hz, 1H), 6.75 (d,J=1.8 Hz, 1H), 6.67 (dd, J=9, 1.8 Hz, 1H), 5.39˜5.34 (m, 2H), 5.31˜5.26(m, 2H), 4.79 (d, J=2.4 Hz, 2H), 4.23 (m, 2H), 3.72 (s, 3H), 2.59 (t,J=2.4 Hz, 1H), 2.07 (s, 3H), 2.06 (s, 3H), 2.05 (s, 3H)

Compound 6

A solution of the compound 5 (930 mg, 1.88 mmol) in isopropyl alcohol (2mL) and chloroform (10 mL) at 0° C. was treated sequentially withsilica-gel (5 g) and NaBH₄ (178 mg, 4.79 mmol). The resulting mixturewas stirred for 3 hours. After the reaction was completed, silica gelwas filtered off. The reaction was extracted with dichloromethane (100mL) and distilled water (100 mL), dried with anhydrous sodium sulfateand concentrated in vacuo. The residue was subjected to columnchromatography to give the compound 6 (610 mg, 65%).

¹H NMR (600 MHz, CDCl₃) δ 7.23 (d, J=8.4 Hz, 1H), 6.72 (d, J=2.4 Hz,1H), 6.61 (dd, 8.4, 2.4 Hz, 1H), 5.35˜5.32 (m, 2H), 5.27 (m, 1H), 5.13(d, J=7.8 Hz, 1H), 4.72 (d, J=2.4 Hz, 2H), 4.63 (d, J=5.4 Hz, 2H), 4.17(m, 1H), 3.73 (s, 3H), 2.07 (s, 3H), 2.05 (s, 3H), 2.04 (s, 3H)

Compound 7

A solution of the compound 6 (250 mg, 0.50 mmol) in dimethylformamide(0.5 mL) was treated with bis(4-nitrophenyl)carbonate (308 mg, 100 mmol)and diisopropylethylamine (DIPEA, 132 μL, 0.75 mmol). The resultingmixture was stirred at room temperature for 3 hours. After the reactionwas completed, the reaction was concentrated under reduced pressure. Theresidue was subjected to column chromatography to give the compound 7(310 mg, 94%).

¹H NMR (600 MHz, CDCl₃) δ 8.26 (d, J=9 Hz, 2H), 7.37 (d, J=9 Hz, 2H),7.34 (d, J=8.4 Hz, 1H), 6.77 (d, J=1.8 Hz, 1H), 6.64 (dd, 7.8, 2.4 Hz,1H), 5.37˜5.33 (m, 2H), 5.30˜5.27 (m, 3H), 5.17 (d, J=7.2 Hz, 1H), 4.74(d, J=2.4 Hz, 2H), 4.18 (m, 1H), 3.74 (s, 3H), 2.54 (t, J=2.4 Hz, 1H),2.07 (s, 3H), 2.05 (s, 3H), 2.04 (s, 3H)

Compound 8

To a solution of the compound 7 (150 mg, 0.227 mmol), MMAF-OMe (169.6mg, 0.227 mmol), and 1-hydroxybenzotriazole anhydrous (HOBt, 6.2 mg,0.0454 mmol) in dimethylformamide (3 mL) were added pyridine (0.8 mL)and diisopropylethylamine (40 μL, 0.227 mmol). The resulting mixture wasstirred at room temperature for 12 hours. After the reaction wascompleted, ethyl acetate (100 mL) and distilled water (100 mL) wereadded. The thus-obtained organic layer was dried with anhydrous sodiumsulfate and concentrated under reduced pressure. The residue wassubjected to column chromatography to give the compound 8 (146 mg, 50%).

EI-MS m/z: 1067 (M⁺)

MMAF-OMe was prepared according to the methods described in U.S.61/483,698, ChemPharmBull, 1995, 43(10), 1706-1718, U.S. Pat. No.7,423,116, U.S. Pat. No. 7,498,298, and WO2002/088172, the contents ofeach of these references are hereby incorporated by reference in theirentirety.

LCB14-0592

A solution of the compound 8 (85 mg, 0.067 mmol) in methanol (2 mL) wastreated at 0° C. with a solution of LiBH₄ (28.2 mg, 0.670 mmol) indistilled water (1 mL). The resulting mixture was stirred at roomtemperature for 3 hours. After the reaction was completed, methanol wasremoved under reduced pressure. The residue was dissolved in distilledwater (50 mL) and acidified with acetic acid to pH=3. The reaction wasextracted three times with dichloromethane (3×50 mL). The combinedorganic layer was concentrated under reduced pressure to give a solidwhich was washed with diethyl ether (50 mL) to yield the compoundLCB14-0592 (62 mg, 83%).

EI-MS m/z: 1112(M⁺)

2-5. Glucuronide Linker-MMAE (LCB14-0598)

Compound 3

A solution of the compound 7 of Example 2-4 (150 mg, 0.227 mmol), MMAE(163 mg, 0.227 mmol; ChemPharmBull, 1995, 43(10), 1706-1718, U.S. Pat.No. 7,423,116, WO2002/088172), and anhydrous 1-hydroxybenzotriazole(HOBt, 6.2 mg, 0.0454 mmol) in dimethylformamide (3 mL) was treated withpyridine (0.8 mL) and diisopropylethylamine (40 μL, 0.227 mmol). Theresulting mixture was stirred at room temperature for 24 hours. Afterthe reaction was completed, the resulting mixture was diluted with ethylacetate (100 mL), 0.5N HCl (10 mL), and distilled water (100 mL). Thethus-obtained organic layer was dried with anhydrous sodium sulfate andconcentrated under reduced pressure. The residue was subjected to columnchromatography to give the compound 3 (30 mg, 10%).

EI-MS m/z: 1238(M⁺)

LCB14-0598

A solution of the compound 3 (30 mg, 0.024 mmol) in methanol (3 mL) wastreated at 0° C. with LiOH (10 mg, 0.24 mmol) in distilled water (0.5mL). The resulting mixture was stirred for 3 hours at room temperature.After the reaction was completed, the organic solvent was removed underreduced pressure. The resulting product was diluted with distilled water(50 mL) and acidified with 0.5N HCl to pH=3. Extraction withdichloromethane (50 mL) followed by concentration under reduced pressuregave the compound LCB14-0598 (21 mg, 79%).

EI-MS m/z: 1098(M⁺)

2-6. Glucuronide Linker-MMAF-Methyl Amide (LCB14-0600)

Compound 2

A solution of the compound 1 (Z-MMAF, 558 mg, 0.644 mmol, ChemPharmBull,1995, 43(10), 1706-1718) in dimethylformamide (5 mL) was treated withmethylamine hydrochloride (130 mg, 1.932 mmol), diethylcyanophosphonate(DEPC, 144 mg, 0.966 mmol), and triethylamine (270 μL, 1.932 mmol). Theresulting mixture was stirred at room temperature for 12 hours. Afterthe reaction was completed, ethyl acetate (100 mL) and distilled water(100 mL) were added. The thus-obtained organic layer was dried withanhydrous sodium sulfate and concentrated under reduced pressure. Theresidue was subjected to column chromatography to give the compound 2(490 mg, 86%).

EI-MS m/z: 879(M⁺)

LCB14-0601 (MMAF-Methyl Amide)

The compound 2 (470 mg, 0.53 mM) was dissolved in tert-butanol (t-BuOH,8 mL) and distilled water (0.8 mL). At 0° C., 10% Pd/C (50 mg) wasadded. The resulting mixture was stirred in H₂ gas for 2 hours. Afterthe reaction was completed, the Pd/C was filtered using celite. Theresulting filtered solution was concentrated under reduced pressure togive the compound LCB14-0601 (340 mg, 85%).

EI-MS m/z: 745(M⁺)

Compound 3

A solution of the compound 7 of Example 2-4 (133 mg, 0.20 mmol),LCB14-601 (150 mg, 0.20 mmol), and anhydrous 1-hydroxybenzotriazole(HOBt, 5.44 mg, 0.04 mmol) in dimethylamide (3 mL) was treated withpyridine (0.8 mL) and diisopropylethylamine (DIPEA, 35 μL, 0.20 mmol).The resulting mixture was stirred at room temperature for 12 hours.After the reaction was completed, ethyl acetate (100 mL) and 0.5N HClsolution (50 mL) were added. The thus-obtained organic layer was driedwith anhydrous sodium sulfate and concentrated under reduced pressure.The residue was subjected to column chromatography to give the compound3 (123 mg, 48%).

EI-MS m/z: 1265(M⁺)

LCB14-0600 (Glucuronide Linker-MMAF-Methyl Amide)

A solution of the compound 3 (60 mg, 0.047 mmol) in methanol (3 mL) wastreated at 0° C. with LiOH (20 mg, 0.47 mmol) in distilled water (0.5mL). The resulting mixture was stirred at room temperature for 2 hours.After the reaction was completed, the organic solvent was removed underreduced pressure. The residue was diluted with distilled water (50 mL)and acidified with 0.5N HCl to pH=3. Extraction with dichloromethane (50mL) followed by concentration gave the compound LCB14-0600 (25 mg, 47%).

EI-MS m/z: 1125(M⁺)

2-7. Azide-Linker-NBD: LCB14-0529

Compound 2

A solution of the compound 1 (4 g, 12.67 mmol) and N-methylmorpholine(1.6 mL, 14.57 mmol) in tetrahydrofuran (30 mL) was treated slowly withisobutylchlroroformate (1.8 mL, 13.94 mmol) under nitrogen atmosphere at−15° C. The resulting mixture was stirred at the same temperature for 30minutes. The resulting mixture was filter-added slowly to a solution ofsodium borohydride (959 mg, 25.34 mmol) in tetrahydrofuran/methanol (36mL/12 mL) at −78° C. with efficient stirring. The reactant was slowlywarmed up to room temperature while being stirred for 2 hours. After thereaction was completed, acetic acid (4 mL) was added and stirred for 15minutes. Ethyl acetate (100 mL) and distilled water (100 mL) were added.The thus-obtained organic layer was dried with anhydrous sodium sulfateand concentrated under reduced pressure. The residue was subjected tocolumn chromatography to give the compound 2 (3.69 g, 96.5%).

¹H NMR (600 MHz, CDCl₃) δ 4.50 (s, 1H), 3.64 (q, J=6.6 Hz, 2H), 3.11 (m,2H), 1.56 (m, 2H), 1.44 (m, 11H), 1.29 (m, 10H)

Compound 3

A solution of the compound 2 (450 mg, 1.73 mmol) and N-methylmorpholine(381 μL, 3.46 mmol) in tetrahydrofuran (5 mL) was treated slowly withmethanesulfonic anhydride (363 mg, 2.07 mmol) under nitrogen atmosphereat 0° C. The resulting mixture was slowly warmed up to room temperaturewhile being stirred for 1 hour. After the reaction was completed, ethylacetate (50 mL) and distilled water (50 mL) were added. Thethus-obtained organic layer was dried with anhydrous sodium sulfate andconcentrated under reduced pressure. The residue was subjected to columnchromatography to give the compound 3 as a white solid (520 mg, 89%).

¹H NMR (600 MHz, CDCl₃) δ 4.50 (s, 1H), 4.22 (t, J=6.6 Hz, 2H), 3.11 (m,2H), 3.01 (s, 3H), 1.74 (m, 2H), 1.44-1.36 (m, 13H), 1.29 (m, 8H)

Compound 4

A solution of the compound 3 (520 mg, 1.54 mmol) in dimethylformamide (5mL) was treated with sodium azide (120 mg, 1.85 mmol) under nitrogenatmosphere and the resulting mixture was stirred at 70° C. for 3 hours.After the reaction was completed, ethyl acetate (50 mL) and distilledwater (50 mL) were added. The thus-obtained organic layer was dried withanhydrous sodium sulfate and concentrated under reduced pressure to givethe compound 4 in liquid form (430 mg, 98%).

¹H NMR (600 MHz, CDCl₃) δ 4.49 (s, 1H), 3.26 (t, J=6.9 Hz, 2H),3.09-3.12 (m, 2H), 1.59 (m, 2H), 1.44 (m, 11H), 1.33 (m, 10H)

Compound 5

A solution of the compound 4 (430 mg, 1.51 mmol) in dichloromethane (6mL) was treated with 4M-HCl in 1,4-dioxane (4 mL) under nitrogenatmosphere at 0° C. The resulting mixture was stirred for 3 hours andconcentrated under reduced pressure to give the compound 5 (330 mg,99%).

¹H NMR (600 MHz, CDCl₃) δ 8.29 (s, 2H), 3.26 (t, J=6.9 Hz, 2H), 2.98 (m,2H), 1.46 (m, 2H), 1.59 (m, 2H), 1.31-1.39 (m, 10H)

LCB14-0529

A solution of the compound 5 (326 mg, 1.47 mmol) in a mixture solvent(10 mL) of acetonitrile and 25 mmol sodium bicarbonate was treated with4-chloro-7-nitrobenzofurazan (442 mg, 2.20 mmol). The resulting mixturewas stirred for 3 hours at room temperature. Ethyl acetate (50 mL) anddistilled water (50 mL) were added. The thus-obtained organic layer wasdried with anhydrous sodium sulfate and concentrated under reducedpressure. The residue was subjected to column chromatography to give thecompound LCB14-0529 (250 mg, 49%).

¹H NMR (600 MHz, CDCl₃) δ 8.48 (d, J=8.4 Hz, 1H), 6.16 (d, 8.4 Hz, 1H),3.47 (q, 6.6 Hz, 2H), 3.24 (t, 6.9 Hz, 2H), 1.79 (m, 2H), 1.59 (m, 2H),1.42-1.48 (m, 2H), 1.20-1.37 (m, 8H)

2-8. Azide-Linker-NBD: LCB14-0530

Compound 2

A solution of tri(ethylene)glycol (5 g, 33.29 mmol) in dichloromethane(30 mL) was treated with p-toluenesulfonyl chloride (13.96 g, 73.24mmol) and potassium hydroxide (8.96 g, 159.79 mmol) under nitrogenatmosphere at 0° C. The resulting mixture was stirred for 3 hours at 0°C. Ethyl acetate (100 mL) and distilled water (100 mL) were added. Thethus-obtained organic layer was dried with anhydrous sodium sulfate andconcentrated under reduced pressure. The residue was subjected to columnchromatography to give the compound 2 (13.2 g, 86.5%) as a white solid.

¹H NMR (600 MHz, CDCl₃) δ 7.79 (m, 4H), 7.35 (m, 4H), 4.14 (m, 4H), 3.65(m, 4H), 3.53 (s, 4H), 2.44 (s, 6H)

Compound 3

A solution of the compound 2 (4.5 g, 9.81 mmol) in dimethylformamide (20mL) was treated with sodium azide (1.6 g, 24.52 mmol) under nitrogenatmosphere. The resulting mixture was stirred at 65° C. for 10 hours.Ethyl acetate (100 mL) and distilled water (100 mL) were added. Thethus-obtained organic layer was dried with anhydrous sodium sulfate andconcentrated under reduced pressure. The residue was subjected to columnchromatography to give the compound 3 (1.96 g, 99%).

¹H NMR (600 MHz, CDCl₃) δ 3.68-3.66 (m, 8H), 3.37 (t, J=4.8 Hz, 4H)

Compound 4

A solution of the compound 3 (500 mg, 2.49 mmol) in 6.6 mL of a mixedsolvent of diethyl ether, tetrahydrofuran, and IN HCl (V:V:V=3:0.6:3). Asolution of triphenylphosphine (655 mg, 2.49 mmol) in diethyl ether (3.5mL) was slowly added over 5 minutes. The resulting mixture was stirredat room temperature for 5 hours. The resulting mixture was diluted withethyl acetate (50 mL) and distilled water (50 mL) and neutralized with1N NaOH solution. The thus-obtained organic layer was dried withanhydrous sodium sulfate and concentrated under reduced pressure. Theresidue was subjected to column chromatography to give the compound 4(370 mg, 85%).

¹H NMR (600 MHz, CDCl₃) δ 3.69-3.63 (m, 6H), 3.52 (t, J=5.1 Hz, 2H),3.40 (t, J=4.8 Hz, 2H), 2.87 (t, J=5.1 Hz, 2H)

LCB14-0530

A solution of the compound 4 (200 mg, 1.14 mmol) in tetrahydrofuran (4mL) was treated sequentially with triethylamine (320 μL, 2.28 mmol) anda solution of 4-chloro-7-nitrobenzofurazan (442 mg, 2.20 mmol) intetrahydrofuran (1 mL). The resulting mixture was stirred at roomtemperature for 1 hour. Ethyl acetate (50 mL) and distilled water (50mL) were added. The thus-obtained organic layer was dried with anhydroussodium sulfate and concentrated under reduced pressure. The residue wassubjected to column chromatography to give the compound LCB14-0530 (305mg, 78.8%).

¹H NMR (600 MHz, CDCl₃) δ 8.47 (d, J=8.4 Hz, 1H), 6.75 (s, 1H), 6.17 (d,J=8.4 Hz, 1H), 3.86 (t, J=4.8 Hz, 2H), 3.66-3.73 (m, 8H), 3.41 (t, J=4.8Hz, 2H)

2-9. Azide-Linker-Drug: LCB14-0505, -0531, and -0510

Compound 2

The compound 1 was prepared with reference to the method described inChemPharmBull, 1995, 43(10), 1706-1718, the contents of which are herebyincorporated by reference in their entirety. A solution of the compound1 (0.50 g, 0.57 mmol) in tert-butanol (6 mL) and water (0.6 mL) wasstirred for 4 hours under hydrogen atmosphere with Pd/C (6 mg, 0.06mmol). The reactant solution was filtered through a celite pad and thefiltrate was concentrated under reduced pressure to give the compound 2(0.42 g) as a white solid.

EI-MS m/z: 747(M⁺)

Compound 9

Chromium(VI) trioxide(CrO₃, 7 g, 0.07 mol) was dissolved in distilledwater (10 mL) at 0° C. To the solution was added sequentially 18M-H₂SO₄(6.1 mL, 0.11 mol) and distilled water (20 mL). The resulting mixturewas stirred for 5 minutes (=Jones reagent). A solution of9-bromo-1-nonanol (5 g, 22.4 mmol) in acetone (250 mL) was treatedslowly with the Jones reagent (18 mL) at −5° C. After stirring theresulting mixture for 3 hours at room temperature, the greenish solidwas filtered off and the filtrate was concentrated. The residue wasextracted with diethyl ether (100 mL) and water (50 mL). The organicextract was dried with anhydrous Na₂SO₄, filtered and concentrated invacuo. The residue was subjected to flash column chromatography to givethe compound 9 (4.95 g, 93%).

¹H NMR (600 MHz, CDCl₃) δ 3.40 (t, J=6.6 Hz, 2H), 2.35 (t, J=7.2 Hz,2H), 1.85 (m, 2H), 1.62 (m, 2H), 1.41 (m, 2H), 1.32 (m, 6H)

Compound 10

A solution of the compound 9 (4 g, 16.86 mmol) in N,N-dimethylformamide(15 mL) was treated with sodium azide (1.64 g, 25.29 mmol). Theresulting mixture was heated to 80° C. for 6 hours with stirring. Afterthe reaction was complete, ethyl acetate (100 mL) and distilled water(100 mL) were added. The thus-obtained organic layer was separated,dried with anhydrous Na₂SO₄, filtered and concentrated in vacuo. Theresidue was subjected to flash column chromatography to give thecompound 10 (3.3 g, 98%).

¹H NMR (600 MHz, CDCl₃) δ 3.26 (t, J=7.2 Hz, 2H), 2.35 (t, J=7.2 Hz,2H), 1.64˜1.57 (m, 4H), 1.35˜1.32 (m, 8H)

LCB14-0505

A solution of the compound 2 (0.16 g, 0.21 mmol) and the9-azido-nonanoic acid (10) (47 mg, 0.24 mmol) in methylene chloride (3mL) was treated with DIPEA (0.06 mL, 0.32 mmol) and PyBOP (0.15 g, 0.28mmol) at 0° C. The resulting mixture was stirred for 3 hours. Theresulting mixture was extracted with methylene chloride (100 mL) andwater (20 mL). The thus-obtained organic layer was concentrated underreduced pressure. The residue was subjected to column chromatographywith ethyl acetate and hexane to give the compound LCB14-0505 (0.12 g,59%) as a white solid.

EI-MS m/z: 928(M⁺)

LCB14-0531

The compound LCB14-0531 (65%) was prepared in a similar method to theabove-described method.

EI-MS m/z: 917(M⁺)

LCB14-0510

The compound 6 was prepared using the methods described inBioconjugateChem. 2002, 13, 855-869 and US2005238649, the contents ofeach of these references are hereby incorporated by reference in theirentirety. A solution of the compound 6 (69 mg, 0.15 mmol) and compound 2(100 mg, 0.13 mmol) in DMF (2 mL) was treated with DIPEA (0.04 mL, 0.2mmol) and PyBOP (0.09 g, 0.17 mmol) at 0° C. The resulting mixture wasstirred for 3 hours. Ethyl acetate (100 mL) and water (30 mL) were usedto extract an organic layer, which was concentrated under reducedpressure. The residue was subjected to column chromatography withmethylene chloride and methanol to give the compound LCB14-0510 (94 mg,64%) as a brown solid.

EI-MS m/z: 1199(M⁺)

2-10. Acetylene-Linker-NBD: LCB14-0532

Compound 2

A solution of the compound 1 (1 g, 5.93 mmol) in 10 mL ofdimethylformamide was treated with sodium azide (578 mg, 8.89 mmol)under nitrogen atmosphere. The resulting mixture was stirred at 80° C.for 3 hours. After the reaction was completed, ethyl acetate (50 mL) anddistilled water (50 mL) were added. The thus-obtained organic layer wasdried with anhydrous sodium sulfate and concentrated under reducedpressure to give the compound 2 (1.03 g, 99%).

¹H NMR (600 MHz, CDCl₃) δ 3.75 (m, 2H), 3.69 (m, 6H), 3.62 (m, 2H), 3.41(t, J=3.5 Hz, 2H), 2.30 (m, 1H)

Compound 3

To a suspension of sodium hydride (55% in mineral oil, 250 mg, 5.7 mmol)in tetrahydrofuran (10 mL) at 0° C. was added a solution of the compound2 (500 mg, 2.85 mmol) in tetrahydrofuran (5 mL). The resulting mixturewas stirred for 1 hour. The resulting mixture was then warmed up to roomtemperature and stirred for 2 hours. Propargyl bromide (80% in toluene,800 μl, 7.12 mmol) was added and the resulting mixture was stirred atroom temperature for 12 hours. Ammonium chloride solution (20 mL) anddiethyl ether (30 mL) were added. The thus-obtained organic layer wasdried with anhydrous sodium sulfate and concentrated under reducedpressure to give the compound 3 (530 mg, 86.6%).

¹H NMR (600 MHz, CDCl₃) δ 4.21 (d, J=2.4 Hz, 2H), 3.66-3.72 (m, 10H),3.39 (t, J=5.1 Hz, 2H), 2.43 (t, J=2.4 Hz, 1H)

Compound 4

A solution of the compound 3 (250 mg, 1.17 mmol) in 3 mL of a mixturesolution of tetrahydrofuran and distilled water (V:V=2:1) was treatedslowly with triphenyl phosphine (461 mg, 1.75 mmol) in tetrahydrofuran(1 mL) over 5 minutes. The resulting mixture was stirred at roomtemperature. After the reaction was completed, diethyl ether (30 mL) anddistilled water (30 mL) were added. The resulting mixture was acidifiedwith 1N HCl, and the organic layer was separated off. The aqueous layerwas diluted with dichloromethane (50 mL) and neutralized with 1N NaOHsolution. The thus-obtained organic layer was separated, dried withanhydrous sodium sulfate and concentrated under reduced pressure to givethe compound 4 (200 mg, 91.3%) in light yellow.

¹H NMR (600 MHz, CDCl₃) δ 4.18 (d, J=2.4 Hz, 2H), 3.59-3.69 (m, 8H),3.48 (t, J=5.4 Hz, 2H), 2.84 (s, 2H), 2.40 (m, 1H)

LCB14-0532

A solution of the compound 4 (195 mg, 1.04 mmol) in tetrahydrofuran (4mL) was treated with triethylamine (290 μL, 2.08 mmol). A solution of4-chloro-7-nitrobenzofurazan (270 mg, 1.35 mmol) in tetrahydrofuran (1mL) was added. The resulting mixture was stirred at room temperature for1 hour. Ethyl acetate (50 mL) and distilled water (50 mL) were added.The thus-obtained organic layer was dried with anhydrous sodium sulfateand concentrated under reduced pressure to give the compound LCB14-0532(280 mg, 77%).

¹H NMR (600 MHz, CDCl₃) δ 8.50 (d, J=8.4 Hz, 1H), 6.99 (s, 1H), 6.19 (d,J=8.4 Hz, 1H), 4.19 (d, J=2.4 Hz, 2H), 3.89 (t, J=5.1 Hz, 2H), 3.68-3.75(m, 10H), 2.41 (t, J=2.4 Hz, 1H)

2-11. Acetylene-Linker-MMAF-OMe (LCB14-0536)

Compound A

To a suspension of NaH (55% in mineral oil, 390 mg, 16.25 mmol) intetrahydrofuran (10 mL) at 0° C. under nitrogen atmosphere was addedslowly a solution of triethylene glycol (4 g, 26.63 mmol) intetrahydrofuran (20 mL). 80% Propargyl bromide in toluene (1.97 g, 13.31mmol) was added slowly. The resulting mixture was stirred at the sametemperature for 2 hours. After the reaction was completed,dichloromethane (100 mL) and water (100 mL) were added. Thethus-obtained organic layer was concentrated and the residue wassubjected to column chromatography to give compound (A) (1 g, 43%) inaqueous form.

¹H NMR (600 MHz, CDCl₃) δ 4.21-4.20 (m, 2H), 3.74-3.66 (m, 10H),3.62-3.61 (m, 2H), 2.43 (t, J=2.4 Hz, 1H)

Compound B

To a solution of the compound A (1 g, 5.31 mmol) in acetone undernitrogen atmosphere at −5° C. was added slowly 5.3 mL of Jones reagent.The resulting mixture, while being slowly warmed up to room temperature,was stirred for 3 hours. After the reaction was completed, ethyl acetate(100 mL) and water (100 mL) were added. The thus-obtained organic layerwas concentrated to give compound (B) (886 mg, 82%) as yellow liquid.

¹H NMR (600 MHz, CDCl₃) δ 4.21 (d, J=2.4, 2H), 4.18-4.17 (m, 2H),3.78-3.77 (m, 2H), 3.74-3.70 (m, 6H), 2.44 (t, J=2.4 Hz, 1H)

LCB14-0536

To a solution of the compound (A) (MMAF-OMe, 100 mg, 0.13 mmol) inacetonitrile (2 mL) at room temperature was added the compound (B) (27mg, 0.13 mmol), PyBOP (104 mg, 0.19 mmol), and DIPEA(0.03 mL, 0.19mmol). The resulting mixture was stirred for 12 hours. After thereaction was completed, ethyl acetate (50 mL) and water (20 mL) wereadded. The thus-obtained organic layer was concentrated under reducedpressure. The residue was subjected to column chromatography withdichloromethane and methanol to give the compound LCB14-0536 (82 mg,68%) as a yellow solid.

EI-MS m/z: 930(M)

2-12. Acetylene-Linker(Peptide Sequence)-MMAF-OMe (LCB14-0589)

Compound 1 (Fmoc-Val-Cit-PAB)

Fmoc-Val-Cit-OH was prepared according to the method described inWO2007/008603, the contents of which are hereby incorporated byreference in their entirety. To a solution of Fmoc-Val-Cit-OH (4.89 g,9.85 mmol) in dichloromethane (50 mL) and methanol (20 mL) undernitrogen atmosphere were added para-aminobenzylalcohol (2.43 g, 19.70mmol) and 1-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (1.98 g, 19.7mmol). The resulting mixture was stirred for 12 hours at roomtemperature. After the reaction was completed, the solvent wasconcentrated. The resulting solid was washed with diethyl ether multipletimes to give the compound 1 (4.12 g, 70%) as a yellow solid.

¹H NMR (600 MHz, DMSO-d₆) δ 10.00 (s, 1H), 8.12 (d, J=7.8 Hz, 1H), 7.89(d, J=7.8 Hz, 2H), 7.75-7.72 (m, 2H), 7.55 (d, J=7.8 Hz, 2H), 7.44-7.41(m, 2H), 7.33-7.31 (m, 2H), 7.23 (d, J=8.4 Hz, 2H), 6.02 (bs, 1H),5.41-5.38 (m, 2H), 5.09 (bs, 1H), 4.42 (bs, 2H), 4.30-4.28 (m, 1H),4.24-4.23 (m, 2H), 3.94-3.91 (m, 1H), 3.02-2.99 (m, 1H), 2.94-2.93 (m,1H), 2.00-1.99 (m, 1H), 1.7 (bs, 1H), 1.60 (bs, 1H), 1.43 (bs, 1H), 1.36(bs, 1H), 0.88-0.84 (m, 6H)

Compound 2 (Fmoc-Val-Cit-PABC-PNA)

A solution of the compound 1 (2 g, 3.32 mmol) in DMF (8 mL) was treatedsequentially with bis(4-nitrophenyl)carbonate (2.02 g, 6.64 mmol) anddiisopropylethylamine (0.647 mL, 4.98 mmol) under nitrogen atmosphere.The resulting mixture was stirred for 12 hours at room temperature.After the reaction was completed, diethyl ether was added forsolidification. The resulting solid was washed with diethyl ether andwater multiple times to give the compound 2 (1.52 g, 60%) as a yellowsolid.

¹H NMR (600 MHz, DMSO-d₆) δ 10.19 (s, 1H), 8.31 (d, J=9.6 Hz, 2H), 8.15(d, J=7.8 Hz, 1H), 7.89 (d, J=7.2 Hz, 2H), 7.75-7.72 (m, 2H), 7.66 (d,J=8.4 Hz, 2H), 7.57 (d, J=9.0 Hz, 2H), 7.43-7.39 (m, 4H), 7.32 (t, J=7.2Hz, 2H), 6.05-6.04 (m, 1H), 5.42 (m, 2H), 5.24 (s, 2H), 4.42 (m, 1H),4.30-4.28 (m, 1H), 425-4.23 (m, 2H), 3.94-3.91 (m, 1H), 3.01-3.00 (m,1H), 2.96-2.94 (m, 1H), 2.00-1.99 (m, 1H), 1.70 (m, 1H), 1.59 (m, 1H),1.45 (m, 1H), 1.37 (m, 1H), 0.89-0.83 (m, 6H).

EI-MS m/z: 767(M⁺)

Compound 3 (Fmoc-Val-Cit-PABC-MMAF-OMe)

A solution of the compound 2 (200 mg, 0.261 mmol) and MMAF-OMe (194 mg,0.261 mmol) in DMF (2 mL) was treated with HOBt (7.1 mg, 0.052 mmol),pyridine (1 mL), and DIPEA (0.045 mL, 0.261 mmol). The resulting mixturewas stirred at room temperature for 12 hours. After the reaction wascompleted, ethyl acetate (30 mL), water (30 mL) and saline solution (30mL) were used to extract an organic layer. The thus-obtained organiclayer was concentrated and subjected to column chromatography to givethe compound 3 (153 mg, 42%) as a yellow solid.

EI-MS m/z: 1375(M⁺)

Compound 4 (Val-Cit-PABC-MMAF-OMe)

To a solution of the compound 3 (153 mg, 0.112 mmol) in tetrahydrofuran(5 mL) at room temperature was added piperidine (0.2 mL). The resultingmixture was stirred at the same temperature for 2 hours. After thereaction was completed, recrystallization was performed with ether andhexane to give the compound 4 (85 mg, 66%) as a light yellow solid.

EI-MS m/z: 1152(M⁺)

LCB14-0589 (Acetylene Linker-Val-Cit-PABC-MMAF-OMe)

To a solution of the compound 4 (85 mg, 0.074 mmol) and the compound Bof Example 2-11 (18 mg, 0.088 mmol) in DMF (2 mL) were added DIPEA (0.03mL, 0.148 mmol) and PyBOP (58 mg, 0.111 mmol). The resulting mixture wasstirred at room temperature for 5 hours. After the reaction wascompleted, extraction was performed with ethyl acetate (20 mL) and water(20 mL). The resulting crude product was subjected to columnchromatography to give the compound LCB14-0589 (35.4 mg, 36%) as a whitesolid.

EI-MS m/z: 1336(M⁺)

2-13. Acetylene-Linker-Val-Cit-PABC-MMAE (LCB14-0602)

Compound 2 (Fmoc-Val-Cit-PABC-MMAE)

To a solution of Fmoc-Val-Cit-PABC-PNP (200 mg, 0.261 mmol) and MMAE(187 mg, 0.261 mmol) in DMF (2 mL) were added HOBt (7.1 mg, 0.052 mmol),pyridine (1 mL), and DIPEA (0.045 mL, 0.261 mmol). The resulting mixturewas stirred at room temperature for 12 hours. After the reaction wascompleted, ethyl acetate (30 mL), water (30 mL), and saline solution (30mL) were used to extract an organic layer. The thus-obtained organiclayer was concentrated and subjected to column chromatography to givethe compound 2 (50 mg, 14.3%) as a yellow solid.

EI-MS m/z: 1346(M⁺)

Compound 3 (Val-Cit-PABC-MMAE)

To a solution of the compound 2 (50 mg, 0.037 mmol) in tetrahydrofuran(5 mL) at room temperature was added piperidine (0.1 mL). The resultingmixture was stirred at the same temperature for 2 hours. After thereaction was completed, recrystallization was performed with ether andhexane to give the compound 3 (37 mg, 89%) as a light yellow solid.

EI-MS m/z: 1124(M⁺)

LCB14-0602 (Acetylene Linker-Val-Cit-PABC-MMAE)

To a solution of the compound 3 (35 mg, 0.031 mmol) and The compound Bof Example 2-11 (7.6 mg, 0.037 mmol) in DMF (2 mL) at room temperaturewere added DIPEA (0.011 mL, 0.062 mmol) and PyBOP (24 mg, 0.47 mmol).The resulting mixture was stirred for 5 hours. After the reaction wascompleted, extraction was performed with ethyl acetate (20 mL) and water(20 mL). The resulting crude product was subjected to columnchromatography to give the compound LCB14-0602 (28.5 mg, 70%) as a whitesolid.

EI-MS m/z: 1308(M⁺)

2-14. Azide Linker-PBD (Pyrrolobenzodiazepin) Dimer (LCB14-0577)

Compound 2

To a solution of the compound 1 (1.22 g, 7.08 mmol), triphenylphosphine(TPP, 2.23 g, 8.50 mmol), and hexaethylene glycol (2 g, 7.08 mmol) intetrahydrofuran (10 mL) at 0° C. under nitrogen atmosphere was addeddiisopropyl azodicarboxylate (DIAD, 1.67 mL, 8.50 mmol). The resultingmixture was stirred for 1 hour. After the reaction was completed, ethylacetate (50 mL) and distilled water (50 mL) were added. Thethus-obtained organic layer was dried with anhydrous sodium sulfate andconcentrated under reduced pressure. The residue was subjected to columnchromatography to give the compound 2 (1.4 g, 45%).

¹H NMR (600 MHz, CDCl₃) δ 7.35 (d, J=8.4 Hz, 2H), 6.80 (d, J=8.4 Hz,2H), 4.09 (t, J=4.8 Hz, 2H), 3.84 (t, J=4.8 Hz, 2H), 3.72 (t, J=4.8 Hz,4H), 3.68˜3.65 (m, 14H), 3.60 (t, J=4.8 Hz, 2H), 2.85 (bs, 1H)

Compound 3

To a solution of the compound 2 (300 mg, 0.68 mmol) in 1,4-dioxane (5mL) were sequentially added potassium acetate (200 mg, 2.04 mmol),PdCl₂(dppf) (28 mg, 0.034 mmol), and bis(pinacolato)diboron (174 mg,0.68 mmol). The resulting mixture was stirred at 70° C. for 12 hours.After the reaction was completed, ethyl acetate (50 mL) and distilledwater (50 mL) were added. The thus-obtained organic layer was dried withanhydrous sodium sulfate and concentrated under reduced pressure. Theresidue was subjected to column chromatography to give the compound 3(300 mg, 90%).

¹H NMR (600 MHz, CDCl₃) δ 7.73 (d, J=8.4 Hz, 2H), 6.90 (d, J=8.4 Hz,2H), 4.15 (t, J=4.8 Hz, 2H), 3.86 (t, J=4.8 Hz, 2H), 3.73˜3.72 (m, 4H),3.68˜3.64 (m, 14H), 3.60 (t, J=4.8 Hz, 2H), 1.33 (s, 12H)

Compound 4

The compound 4 was prepared according to the methods described inWO2006/111759 A1, WO2010/043880 A1, and WO2010/010347 A1, the contentsof each of these references are hereby incorporated by reference intheir entirety.

¹H NMR (600 MHz, CDCl₃) δ 7.35 (s, 1H), 7.29 (d, J=9 Hz, 2H), 7.27 (s,1H), 7.23 (s, 1H), 7.17 (s, 1H), 6.89 (d, J=9 Hz, 2H), 6.77 (s, 1H),6.75 (s, 1H), 5.91 (m, 2H), 5.23 (d, J=9 Hz, 2H), 5.21 (d, J=9 Hz, 2H),4.29 (m, 2H), 4.17˜4.13 (m, 4H), 3.96˜3.91 (m, 8H), 3.82 (s, 3H), 3.33(m, 2H), 2.82 (m, 2H), 2.44 (m, 2H), 0.90 (2s, 18H), 0.27 (2s, 12H)

Compound 5

The compound 4 (83 mg, 0.059 mmol), sodium carbonate (10 mg, 0.089mmol), and Pd(TPP)₄ (3.4 mg, 0.003 mmol) were sequentially dissolved ina mixture solvent of ethanol/toluene/distilled water (0.3 mL/0.3 mL/0.3mL). A solution of the compound 3 (31.6 mg, 0.065 mmol) in toluene (3mL) was added. The resulting mixture was stirred at room temperature for1 hour. After the reaction was completed, ethyl acetate (50 mL) anddistilled water (50 mL) were added. The thus-obtained organic layer wasdried with anhydrous sodium sulfate and concentrated under reducedpressure. The residue was subjected to column chromatography to give thecompound 5 (79 mg, 74%).

¹H NMR (600 MHz, CDCl₃) δ 7.35 (m 2H), 7.31˜7.27 (m, 6H), 6.92˜6.89 (m,4H), 6.78 (s, 2H), 5.90 (d, J=9 Hz, 2H), 5.23 (d, J=12.6 Hz, 2H), 4.30(m, 2H), 4.16˜4.13 (m, 6H), 3.97˜3.94 (m, 8H), 3.87 (t, J=4.8 Hz, 2H),3.83 (s, 3H), 3.74˜3.64 (m, 18H), 3.61 (m, 2H), 3.34 (m 2H), 2.82 (m,2H), 2.45 (m, 2H), 0.90 (s, 18H), 0.25 (2s, 12H)

Compound 6

To a solution of the compound 5 (250 mg, 0.155 mmol) in tetrahydrofuran(3 mL) at 0° C. were added 4-methylmorpholine (34.2 μL, 0.310 mmol) andmethane sulfonic anhydride (Ms₂O, 32.5 mg, 0.186 mmol). The resultingmixture was stirred at room temperature for 3 hours. After the reactionwas completed, ethyl acetate (50 mL) and distilled water (50 mL) wereadded. The thus-obtained organic layer was dried with anhydrous sodiumsulfate and concentrated under reduced pressure. The residue wassubjected to column chromatography to give the compound 6 (220 mg, 84%).

¹H NMR (600 MHz, CDCl₃) δ 7.33 (m, 2H), 7.28˜7.23 (m, 6H), 6.89˜6.86 (m,4H), 6.76 (s, 2H), 5.88 (d, J=9 Hz, 2H), 5.21 (d, J=12.6 Hz, 2H), 4.35(m, 2H), 4.26 (m, 2H), 4.13˜4.11 (m 6H), 3.92 (s, 6H), 3.84 (t, J=4.8Hz, 2H), 3.80 (s, 3H), 3.74˜3.60 (m, 20H), 3.31 (m, 2H), 3.06 (s, 3H),2.80 (m, 2H), 2.43 (m, 2H), 0.88 (s, 18H), 0.23 (2s, 12H)

Compound 7

To a solution of the compound 6 (100 mg, 0.059 mmol) indimethylformamide (2 mL) was added sodium azide (NaN₃, 4.6 mg, 0.071mmol). The resulting mixture was stirred at 55° C. for 4 hours. Afterthe reaction was completed, ethyl acetate (50 mL) and distilled water(50 mL) were added. The thus-obtained organic layer was dried withanhydrous sodium sulfate and concentrated under reduced pressure. Theresidue was subjected to column chromatography to give the compound 7(85 mg, 88%).

¹H NMR (600 MHz, CDCl₃) δ 7.33 (bs, 2H), 7.28˜7.24 (m, 6H), 6.89˜6.87(m, 4H), 6.76 (s, 2H), 5.88 (d, J=9 Hz, 2H), 5.21 (d, J=12.6 Hz, 2H),4.26 (m, 2H), 4.13˜4.11 (m, 6H), 3.92 (m, 8H), 3.84 (t, J=4.8 Hz, 2H),3.80 (s, 3H), 3.71 (m, 2H), 3.67˜3.64 (m, 16H), 3.36 (t, J=4.8 Hz, 2H),3.31 (m, 2H), 2.80 (m, 2H), 2.43 (m, 2H), 0.88 (s, 18H), 0.23 (2s, 12H)

LCB14-0577

To a solution of the compound 7 (80 mg, 0.049 mmol) in tetrahydrofuran(1.5 mL) were added 1N-ammonium acetate (1 mL) and 10% cadmium/leadcouple (120 mg). The resulting mixture was stirred at room temperaturefor 4 hours. After the reaction was completed, dichloromethane (50 mL)and distilled water (50 mL) were added. The thus-obtained organic layerwas dried with anhydrous sodium sulfate and concentrated under reducedpressure. The residue was subjected to column chromatography to give thecompound LCB14-0577 (9 mg, 18%).

¹H NMR (600 MHz, CDCl₃) δ 7.86 (d, J=4.2 Hz, 2H), 7.36 (m, 2H),7.31˜7.23 (m, 6H), 6.89˜6.80 (m, 6H), 4.34˜4.22 (m, 6H), 4.11 (m, 2H),3.92 (m, 6H), 3.84˜3.77 (m, 5H), 3.71 (m, 2H), 3.67˜3.63 (, 18H), 3.36(m, 2H), 3.03 (m, 2H), 2.44˜2.40 (m, 2H)

EI-MS m/z: 1017(M⁺)

2-15. Acetylene-Linker-PBD Dimer (LCB14-0578)

Compound 2

To a solution of the compound 6 of Example 2-14 (95 mg, 0.056 mmol) inacetonitrile (1 mL) was added a solution of sodium carbonate (18 mg,0.168 mmol) in propargyl amine (18 μL, 0.28 mmol) and distilled water(500 μL). The resulting mixture was stirred at 40° C. for 12 hours.After the reaction was completed, ethyl acetate (50 mL) and distilledwater (50 mL) were added. The thus-obtained organic layer was dried withanhydrous sodium sulfate and concentrated under reduced pressure. Theresidue was subjected to column chromatography to give the compound 2(45 mg, 48%).

¹H NMR (600 MHz, CDCl₃) δ 7.35 (m, 2H), 7.30˜7.27 (m, 6H), 6.91˜6.89 (m,4H), 6.78 (s, 2H), 5.91 (d, J=9 Hz, 2H), 5.23 (d, J=11.4 Hz, 2H), 4.30(m, 2H), 4.16˜4.11 (m, 6H), 3.94 (s, 6H), 3.87 (t, J=4.8 Hz, 2H), 3.83(s, 3H), 3.73 (m, 2H), 3.69˜3.60 (m 18H), 3.45 (d, J=2.4 Hz, 2H), 3.33(m, 2H), 2.87 (t, J=4.8 Hz, 2H), 2.82 (m, 2H), 2.45 (m, 2H), 2.22 (t,J=4.4 Hz, 1H), 0.90 (s, 18H), 0.24 (2s, 12H)

LCB14-0578

To a solution of the compound 2 (40 mg, 0.024 mmol) in tetrahydrofuran(750 μL) were added 1N-ammonium acetate (0.5 mL) and 10% cadmium/leadcouple (70 mg). The resulting mixture was stirred at room temperaturefor 4 hours. After the reaction was completed, dichloromethane (50 mL)and distilled water (50 mL) were added. The thus-obtained organic layerwas dried with anhydrous sodium sulfate and concentrated under reducedpressure. The residue was subjected to column chromatography to give thecompound LCB14-0578 (13 mg, 52%).

¹H NMR (600 MHz, CDCl₃) δ 7.88 (d, J=4.2 Hz, 2H), 7.38 (m, 2H),7.33˜7.28 (m, 6H), 6.91˜6.86 (m, 6H), 4.38˜4.20 (m, 6H), 4.13 (m 2H),3.94 (s, 6H), 3.88˜3.80 (m, 5H), 3.73 (m, 2H), 3.69˜3.61 (m, 16H), 3.46(d, J=2.4 Hz, 2H), 3.39 (m, 2H), 3.30 (m, 2H), 2.88 (t, J=4.8 Hz, 2H),2.43 (m, 2H), 2.23 (t, J=4.4 Hz, 1H))

EI-MS m/z: 1028(M⁺)

2-16. Acetylene-Linker-PBD Dimer (Pyridine Version) (LCB14-0582)

Compound 2

To a suspension of NaH (55% in mineral oil, 184 mg, 4.22 mmol) intetrahydrofuran (5 mL) at 0° C. under nitrogen atmosphere was addedhexaethyleneglycol (2.4 g, 8.44 mmol) in tetrahydrofuran (3 mL). Theresulting mixture was stirred for 10 minutes at 0° C. A mixture solutionprepared by dissolving the compound 1 (1 g, 4.22 mmol) indimethylformamide (0.5 mL) and tetrahydrofuran (0.5 mL) was slowlyadded. The resulting mixture was stirred at room temperature for 1 hourand then stirred at 70° C. for 12 hours. After cooling the resultingmixture to 0° C., distilled water (2 mL) was added. After the reactionwas completed, ethyl acetate (100 mL) and distilled water (100 mL) wereadded. The thus-obtained organic layer was dried with anhydrous sodiumsulfate and concentrated under reduced pressure. The residue wassubjected to column chromatography to give the compound 2 (1.5 g, 81%).

¹H NMR (600 MHz, CDCl₃) δ 8.13 (d, J=2.4 Hz, 1H), 7.61 (dd, J=8.4, 2.4Hz, 1H), 6.67 (d, J=9 Hz, 1H), 4.41 (m, 2H), 3.81 (m, 2H), 3.70˜3.61 (m,18H), 3.58 (m, 2H), 2.71 (bs, 1H)

Compound 3

A solution of the compound 2 (500 mg, 1.14 mmol) in dimethylformamide (5mL) was treated sequentially with potassium acetate (336 mg, 3.42 mmol),PdCl₂(dppf) (46.5 mg, 0.057 mmol), and bis(pinacolato)diboron (318 mg,1.25 mmol). The resulting mixture was stirred at 70° C. for 12 hours.After the reaction was completed, ethyl acetate (100 mL) and distilledwater (50 mL) were added. The thus-obtained organic layer was dried withanhydrous sodium sulfate and concentrated under reduced pressure. Theresidue was subjected to column chromatography to give the compound 3(250 mg, 45%).

¹H NMR (400 MHz, CDCl₃) δ 8.50 (s, 1H), 7.90 (d, J=8.4 Hz, 1H), 6.74 (d,J=8.4 Hz, 1H), 4.50 (t, J=4.8 Hz, 2H), 3.84 (m, 2H), 3.74˜3.70 (m, 20H),1.33 (s, 12H)

Compound 5

The compound 4 (245 mg, 0.175 mmol), sodium carbonate (28 mg, 0.262mmol), and Pd(TPP)₄ (10 mg, 0.009 mmol) were sequentially dissolved in amixture solution of ethanol/toluene/distilled water (1.5 mL/1.5 mL/1.5mL). A solution of the compound 3 (94 mg, 0.192 mmol) in toluene (1.5mL) was added. The resulting mixture was stirred at room temperature for12 hours. After the reaction was completed, ethyl acetate (100 mL) anddistilled water (100 mL) were added. The thus-obtained organic layer wasdried with anhydrous sodium sulfate and concentrated under reducedpressure. The residue was subjected to column chromatography to give thecompound 5 (100 mg, 35.5%).

¹H NMR (600 MHz, CDCl₃) δ 8.02 (d, J=2.4 Hz, 1H), 7.66 (m, 1H), 7.38 (s,1H), 7.35 (s, 1H), 7.29 (d, J=9 Hz, 2H), 7.27 (m, 2H), 6.89 (d, J=9 Hz,2H), 6.80 (d, J=8.4 Hz, 1H), 6.78 (s, 2H), 5.90 (d, J=9 Hz, 2H), 5.23(dd, J=11.4, 4.2 Hz, 2H), 4.47 (m, 2H), 4.29 (m, 2H), 4.17˜4.12 (m, 2H),3.4 (m, 8H), 3.86 (t, J=4.8 Hz, 2H), 3.82 (m, 4H), 3.74˜3.65 (m, 18H),3.61 (m, 2H), 3.33 (m, 2H), 2.83 (m, 2H), 2.45 (m, 2H), 0.90 (s, 18H),0.25 (2s, 12H)

Compound 6

To a solution of the compound 5 (180 mg, 0.11 mM) in tetrahydrofuran (3ml) at 0° C. were added 4-methylmorpholine (NMM, 61.5 μL, 0.55 mM) andmethane sulfonic anhydride (Ms₂O, 22 mg, 0.121 mM). The resultingmixture was stirred at room temperature for 3 hours. After the reactionwas completed, ethyl acetate (50 ml) and distilled water (50 ml) wereadded to extract an organic layer. The organic layer was dried withanhydride sodium sulfate and concentrated under reduced pressure. Theresidue was subjected to column chromatography to prepare the compound 6(80 mg, 43%).

¹H NMR (600 MHz, CDCl₃) δ 8.03 (d, J=2.4 Hz, 1H), 7.66 (dd, J=7.8, 2.4Hz, 1H), 7.38 (s, 1H), 7.35 (s, 1H), 7.30 (d, J=9 Hz, 2H), 7.27 (m, 2H),6.89 (d, J=9 Hz, 2H), 6.80 (d, J=9 Hz, 1H), 6.78 (s, 2H), 5.90 (d, J=9Hz, 2H), 5.22 (dd, J=12, 4.2 Hz, 2H), 4.47 (m, 2H), 4.38 (m, 2H), 4.30(m, 2H), 4.15 (m, 3H), 3.99˜3.93 (m, 7H), 3.86 (m, 2H), 3.83 (s, 3H),3.76 (m, 2H), 3.71 (m, 2H), 3.69˜3.63 (m, 16H), 3.34 (m, 2H), 3.08 (s,3H), 2.83 (m, 2H), 2.45 (m, 2H), 0.90 (2s, 18H), 0.25 (2s, 12H)

Compound 7

To a solution of the compound 6 (80 mg, 0.047 mmol) in acetonitrile (4mL) was added a solution of sodium carbonate (20 mg, 0.141 mmol) inpropargylamine (30 μL, 0.47 mmol) and distilled water (500 μL). Theresulting mixture was stirred at 50° C. for 12 hours. After the reactionwas completed, ethyl acetate (50 mL) and distilled water (50 mL) wereadded. The thus-obtained organic layer was dried with anhydrous sodiumsulfate and concentrated under reduced pressure. The residue wassubjected to column chromatography to give the compound 7 (25 mg, 32%).

¹H NMR (600 MHz, CDCl₃) δ 8.03 (d, J=1.8 Hz, 1H), 7.66 (dd, J=8.4, 2.4Hz, 1H), 7.38 (s, 1H), 7.35 (s, 1H), 7.30 (d, J=8.4 Hz, 2H), 7.28 (m,2H), 6.89 (d, J=9 Hz, 2H), 6.79 (d, J=9 Hz, 1H), 6.78 (s, 2H), 5.90 (d,J=9 Hz, 2H), 5.22 (dd, J=12, 4.2 Hz, 2H), 4.47 (m, 2H), 4.30 (m, 2H),4.17˜4.14 (m, 3H), 3.98˜3.93 (m, 7H), 3.86 (m, 2H), 3.82 (s, 3H), 3.72(m, 2H), 3.69˜3.60 (m, 18H), 3.45 (d, J=2.4 Hz, 2H), 3.34 (m, 2H), 2.87(t, J=4.8 Hz, 2H), 2.83 (m, 2H), 2.45 (m, 2H), 2.22 (m, 1H), 0.90 (2s,18H), 0.25 (2s, 12H)

LCB14-0582

To a solution of the compound 7 (25 mg, 0.015 mmol) in tetrahydrofuran(750 μL) were added 1N-ammonium acetate (0.5 mL) and 10% cadmium/leadcouple (50 mg). The resulting mixture was stirred at room temperaturefor 3 hours. After the reaction was completed, dimethylchloromethane (50mL) and distilled water (50 mL) were added. The thus-obtained organiclayer was dried with anhydrous sodium sulfate and concentrated underreduced pressure. The residue was subjected to column chromatography togive the compound LCB14-0582 (6 mg, 38.4%).

¹H NMR (600 MHz, CDCl₃) δ 8.00 (m, 1H), 7.88 (m, 2H), 7.60 (m, 1H),7.41˜7.28 (m, 6H), 6.90˜6.71 (m, 5H), 4.46 (m, 2H), 4.35˜4.24 (m, 4H),3.95˜3.79 (m, 11H), 3.70 (m, 2H), 3.68˜3.61 (m, 18H), 3.47 (m, 2H), 3.38(m, 2H), 3.04 (m, 2H), 2.89 (t, J=5.4 Hz, 2H), 2.40 (m, 2H), 2.23 (bs,1H)

EI-MS m/z: 1029(M⁺)

2-17. Amino-Peg5-PBD Dimer (LCB14-0594)

Compound 1

To a solution of the compound 5 of Example 2-14 (456 mg, 0.284 mmol) intetrahydrofuran were added triphenylphosphine (108 mg, 0.411 mmol) andphthalimide (50 mg, 0.341 mmol). DIAD (0.058 mL, 0.340 mmol) was slowlyadded at 0° C. The resulting mixture was stirred at room temperature for2 hours. After the reaction was completed, extraction was performed withdichloromethane (40 mL) and water (40 mL). The residue was subjected tocolumn chromatography to give the compound 1 (492 mg, quantitative) as ayellow solid.

¹H NMR (600 MHz, CDCl₃) δ 7.84-7.82 (m, 2H), 7.70-7.69 (m, 2H), 7.34 (m,2H), 7.29-7.25 (m, 6H), 6.90 (d, J=7.2, 4H), 6.78 (s, 2H), 5.92 (d,J=9.0, 2H), 5.21 (d, J=12.6, 2H), 4.28 (m, 2H), 4.19-4.10 (m, 4H), 3.93(m, 6H), 3.89-3.87 (m, 2H), 3.86-3.84 (m, 2H), 3.82 (s, 3H), 3.74-3.71(m, 4H), 3.67-3.66 (m, 2H), 3.63-3.62 (m, 6H), 3.59-3.58 (m, 6H), 3.33(m, 2H), 2.85-2.82 (m, 2H), 2.42 (m, 2H), 0.91 (s, 18H), 0.27 (2s, 12H)

Compound 2

To a solution of the compound 1 (492 mg, 0.283 mmol) in ethyl alcohol (2mL) and tetrahydrofuran (2 mL) was added hydrazine monohydrate (0.07 mL,1.417 mmol). The resulting mixture was stirred at 60° C. for 5 hours.After the reaction was completed, 2 mL of ethyl acetate was added. Solidwas filtered off. The filtrate was concentrated and subjected to columnchromatography to give the compound 2 (380 mg, 83%) as a yellow solid.

¹H NMR (600 MHz, CDCl₃) δ 7.35 (bs, 2H), 7.29-7.26 (m, 6H), 6.92-6.88(m, 4H), 6.79 (bs, 2H), 5.92 (d, J=8.4, 2H), 5.21 (d, J=12, 2H),4.29-4.28 (m, 2H), 4.19-4.17 (m, 6H), 3.93-3.90 (m, 6H), 3.89-3.87 (m,2H), 3.82 (s, 3H), 3.75-3.73 (m, 2H), 3.69-3.63 (m, 12H), 3.35-3.31 (m,2H), 2.96 (bs, 2H), 2.85 (d, J=16.8, 2H), 2.43 (m, 2H), 0.91 (s, 18H),0.27 (2s, 12H).

EI-MS m/z: 1606(M⁺)

LCB14-0594

A solution of the compound 2 (25 mg, 0.015 mmol) in tetrahydrofuran (1mL) at room temperature was added 1N ammonium acetate (0.4 mL) and 10%Cadmium/lead couple (40 mg). The resulting mixture was stirred at thesame temperature for 12 hours. After the reaction was completed, theresulting mixture was filtered with dichloromethane. The filteredsolution was concentrated and subjected to column chromatography to givethe LCB14-0594 (4 mg, 26%) as a yellow solid.

EI-MS m/z: 990(M⁺)

2-18. Glucuronide-Linker-PBD Monomer (LCB14-0596)

Compound (B)

To a solution of the compound (A) (300 mg, 0.57 mmol) in tetrahydrofuran(5 mL) at room temperature were added N-methylmorpholine (0.16 mL, 1.43mmol) and methanesulfonic anhydride (120 mg, 0.69 mmol). The resultingmixture was stirred for 4 hours. Ethyl acetate (100 mL) and water (50mL) were added. The thus-obtained organic layer was concentrated underreduced pressure. The residue was subjected to column chromatographywith ethyl acetate and hexane to give the compound (B) (330 mg, 96%).

¹H NMR (600 MHz, CDCl₃) δ: 7.53 (s, 1H), 7.40 (s, 1H), 7.39-7.37 (m,2H), 7.33 (t, J=1.8 Hz, 1H), 6.90-6.89 (m, 2H), 5.47 (d, J=10.2 Hz, 1H),4.81 (d, J=10.2 Hz, 1H), 4.62 (dd, J=7.2, 3.0 Hz, 1H), 4.49-4.41 (m,2H), 3.97-3.93 (m, 1H), 3.92 (s, 3H), 3.83 (s, 3H), 3.76-3.72 (m, 1H),3.68-3.64 (m, 1H), 3.17-3.10 (m, 3H), 2.96 (s, 3H), 0.98 (t, J=8.4 Hz,2H), 0.02 (s, 9H).

EI-MS m/z: 603(M⁺)

Compound (C)

To a solution of the compound (B) (330 mg, 0.55 mmol) in DMF (3 mL) atroom temperature was added sodium azide (43 mg, 0.66 mmol). Theresulting mixture was stirred at 60° C. for 3 hours. Ethyl acetate (100mL) and water (50 mL) were added. The thus-obtained organic layer wasconcentrated under reduced pressure. The residue was subjected to columnchromatography with ethyl acetate and hexane to give the compound (C)(307 mg, 99%) as a yellow solid.

¹H NMR (600 MHz, CDCl₃) δ: 7.54 (s, 1H), 7.38-7.37 (m, 3H), 7.34 (t,J=1.8 Hz, 1H), 6.90-6.88 (m, 2H), 5.49 (d, J=10.2 Hz, 1H), 4.76 (d,J=10.2 Hz, 1H), 4.63 (dd, J=7.2, 3.0 Hz, 1H), 3.96-3.93 (m, 1H), 3.92(s, 3H), 3.83 (s, 3H), 3.79-3.75 (m, 1H), 3.69-3.65 (m, 1H), 3.52-3.50(m, 2H), 3.16-3.12 (m, 1H), 3.03-2.99 (m, 1H), 2.96-2.91 (m, 1H), 0.99(t, J=8.4 Hz, 2H), 0.02 (s, 9H).

EI-MS m/z: 550(M⁺)

Compound (1-1)

To a solution of the compound (C) (500 mg, 0.91 mmol) in tetrahydrofuran(2 mL) and distilled water (0.5 mL) at room temperature was addedtriphenylphosphine (285 mg, 1.09 mmol). The resulting mixture wasstirred at 40° C. for 13 hours. Ethyl acetate (200 mL) and water (100mL) were added. The thus-obtained organic layer was concentrated underreduced pressure. The residue was subjected to column chromatographywith ethyl acetate and hexane to give the compound (1-1) (435 mg, 93%)as a yellow solid.

¹H NMR (600 MHz, CDCl₃) δ: 7.50 (s, 1H), 7.38-7.36 (m, 3H), 7.33 (t,J=1.8 Hz, 1H), 6.90-6.88 (m, 2H), 5.47 (d, J=9.6 Hz, 1H), 4.81 (d, J=9.6Hz, 1H), 4.67 (dd, J=7.2, 3.0 Hz, 1H), 3.95-3.92 (m, 1H), 3.91 (s, 3H),3.83 (s, 3H), 3.76-3.72 (m, 1H), 3.68-3.64 (m, 1H), 3.15-3.10 (m, 2H),3.06-2.96 (m, 2H), 2.94-2.88 (m, 1H), 2.86-2.80 (m, 1H), 0.98 (t, J=8.4Hz, 2H), 0.02 (s, 9H).

EI-MS m/z: 524(M⁺)

Compound 3

To a solution of the compound 7 of Example 2-4 (126 mg, 0.190 mmol) andthe compound (1-1) (100 mg, 0.190 mmol) in dimethylformamide (3 mL) wasadded triethylamine (TEA, 80L, 0.57 mmol). The resulting mixture wasstirred at room temperature for 3 hours. After the reaction wascompleted, ethyl acetate (100 mL) and distilled water (100 mL) wereadded. The thus-obtained organic layer was dried with anhydrous sodiumsulfate and concentrated under reduced pressure. The residue wassubjected to column chromatography to give the compound 3 (178 mg, 89%).

¹H NMR (400 MHz, CDCl₃) δ 7.46 (s, 1H), 7.37 (d, J=8.8 Hz, 2H), 7.34 (m,2H), 7.22 (m, 1H), 6.87 (d, J=8.8 Hz, 2H), 6.71 (d, J=2.0 Hz, 1H), 6.60(m, 1H), 5.44 (d, J=10.4 Hz, 1H), 5.34 (m, 2H), 5.27 (m, 1H), 5.16 (d,J=7.6 Hz, 1H), 5.07 (s, 2H), 4.82˜4.77 (m, 2H), 4.68 (d, J=2.0 Hz, 2H),4.60 (m, 1H), 4.19 (d, J=9.2 Hz, 1H), 3.93 (m, 1H), 3.87 (s, 3H), 3.82(s, 3H), 3.72˜3.61 (m, 5H), 3.45 (m, 2H), 3.11 (m, 1H), 2.93˜2.84 (m,2H), 2.51 (bs, 1H), 2.05 (s, 3H), 2.04 (s, 3H), 2.03 (s, 3H), 0.97 (t,J=7.2 Hz, 2H), 0.01 (s, 9H)

Compound 4

To a solution of the compound 3 (100 mg, 0.094 mmol) in methanol (5 mL)at 0° C. was added lithium hydroxide (40 mg, 1.880 mmol) in distilledwater (2 mL). The resulting mixture was stirred at room temperature for3 hours. After the reaction was completed, methanol was removed underreduced pressure. The residue was diluted with distilled water (50 mL)and acidified slowly with acetic acid to pH=3. Extraction was performedthree times with dichloromethane (3×50 mL). The resulting product wasconcentrated under reduced pressure to yield a solid compound. The solidcompound was washed with diethyl ether (50 mL) to give the compound 4(86.5 mg, 100%).

¹H NMR (600 MHz, CD₃OD) δ 7.43 (s, 1H), 7.41 (d, J=9 Hz, 2H), 7.30 (d,J=10.2 Hz, 2H), 7.14 (d, J=7.8 Hz, 1H), 6.90 (d, J=9 Hz, 2H), 6.86 (m,1H), 6.66 (m, 1H), 5.22 (m, 2H), 4.98˜4.94 (m, 3H), 4.71˜4.67 (m, 3H),3.96 (m, 1H), 3.87 (s, 3H), 3.78 (s, 3H), 3.75 (m, 1H), 3.59˜3.47 (m,5H), 3.36 (m, 2H), 3.25 (m, 1H), 3.13 (m, 1H), 2.90 (bs, 1H), 2.85 (m,2H), 0.83 (m, 2H), 0.01 (s, 9H)

EI-MS m/z: 904(M⁺)

LCB14-0596

To a solution of the compound 4 (86.5 mg, 0.094 mmol) in tetrahydrofuran(1 mL) and ethanol (1 mL) at 0° C. was added lithium borohydride2M-tetrahydrofuran solution (940 μL, 1.88 mmol). The resulting mixturewas stirred at room temperature for 12 hours. Additional lithiumborohydride 2M-tetrahydrofuran solution (1.41 mL, 2.82 mmol) was added.The resulting mixture was stirred for 5 hours and cooled to 0° C. Thereaction was quenched by addition of 1% formic acid solution (33 mL).The resulting mixture was stirred for 3 hours. After the reaction wascompleted, extraction was performed with distilled water (50 mL) and amixture solution of ethyl acetate (20 mL) and methanol (10 mL). Theresidue was subjected to column chromatography usingchloroform/methanol/formic acid (V:V:V=9:1:0.05) to give the compoundLCB14-0596 (50 mg, 69%).

EI-MS m/z: 756(M⁺)

2-19. Glucuronide Linker-PBD Dimer (LCB14-0597)

Compound 3

To a solution of the compound 7 of Example 2-4 (150 mg, 0.220 mmol) andthe compound 2 of Example 2-17 (365 mg, 0.220 mmol) in dimethylformamide(3 mL) was added triethylamine (95 μL, 0.66 mmol). The resulting mixturewas stirred at room temperature for 2 hours. After the reaction wascompleted, ethyl acetate (100 mL) and distilled water (100 mL) wereadded. The thus-obtained organic layer was dried with anhydrous sodiumsulfate and concentrated under reduced pressure. The residue wassubjected to column chromatography to give the compound 3 (310 mg, 64%).

¹H NMR (600 MHz, CDCl₃) δ 7.35 (m, 2H), 7.30˜7.25 (m, 7H), 6.90 (m, 4H),6.78 (s, 2H), 6.73 (d, J=2.4 Hz, 1H), 6.60 (dd, 8.4, 1.8 Hz, 1H), 5.90(d, J=2.4 Hz, 2H), 5.36˜5.32 (m, 2H), 5.27 (m, 2H), 5.22 (m, 2H), 5.13(d, J=7.2 Hz, 1H), 5.09 (s, 2H), 4.69 (d, J=2.4 Hz, 2H), 4.29 (m 2H),4.17˜4.13 (m, 6H), 3.94 (m, 8H), 3.85 (t, J=4.8 Hz, 2H), 3.82 (s, 3H),3.73 (s, 3H), 3.71 (m, 2H), 3.67˜3.59 (m, 14H), 3.54 (t, J=4.8 Hz, 2H),3.39˜3.31 (m, 4H), 2.82 (m, 2H), 2.52 (t, J=2.4 Hz, 1H), 2.44 (m, 2H),2.06 (s, 3H), 2.05 (s, 3H), 2.04 (s, 3H), 0.91 (s, 18H), 0.26 (2s, 12H)

Compound 4

To a solution of the compound 3 (100 mg, 0.047 mmol) in methanol (3 mL)and tetrahydrofuran (1.5 mL) at 0° C. was added lithium hydroxide (20mg, 0.47 mmol) in distilled water (1.5 mL). The resulting mixture wasstirred at room temperature for 3 hours. After the reaction wascompleted, organic solvent was removed under reduced pressure. Theresidue was diluted with distilled water (50 mL) and acidified slowlywith 0.5N HCl solution to pH=3. Extraction was performed three timeswith dichloromethane (3×50 mL). The extract was concentrated underreduced pressure to give the compound 4 (93.4 mg, 100%).

¹H NMR (600 MHz, CDCl₃) δ 7.35 (m, 2H), 7.30˜7.24 (m, 7H), 4.89 (m, 4H),6.78 (m, 3H), 6.64 (m, 1H), 5.91 (m, 2H), 5.65 (m, 1H), 5.21 (m, 2H),5.07 (m, 2H), 4.89 (m, 1H), 4.67 (m, 2H), 4.28 (m, 2H), 4.18˜4.12 (m,6H), 3.93 (m, 8H), 3.85˜3.82 (m, 5H), 3.72 (m, 2H), 3.65˜3.54 (m, 20H),3.34˜3.32 (m, 4H), 2.82 (m, 2H), 2.56 (m, 1H), 2.44 (m, 2H), 0.90 (2s,18H), 0.25 (2s, 12H)

LCB14-0597

To a solution of the compound 4 (90 mg, 0.045 mmol) in tetrahydrofuran(1.5 mL) were added 1N-ammonium acetate (1.2 mL) and 10% cadmium/leadcouple (120 mg). The resulting mixture was stirred at room temperaturefor 3 hours. After the reaction was completed, ethyl acetate (50 mL) anddistilled water (50 mL) were added. The thus-obtained organic layer wasdried with anhydrous sodium sulfate and concentrated under reducedpressure. The residue was subjected to column chromatography to give thecompound LCB14-0597 (16.4 mg, 26%).

EI-MS m/z: 1371(M⁺)

2-20. Amino-Peg1-PBD Dimer (LCB14-0599)

Compound 1

To a solution of 4-bromophenol (4.0 g, 23.1 mmol) in ethanol (18 mL) atroom temperature were added sodium hydroxide (1.0 g, 25.40 mmol) and2-bromoethanol (1.7 mL, 23.10 mmol). Ethyl acetate (500 mL) and water(200 mL) were added. The thus-obtained organic layer was concentratedunder reduced pressure. The residue was subjected to columnchromatography with ethyl acetate and hexane to give the compound 1 (4.3g, 86%) in liquid form.

¹H NMR (600 MHz, CDCl₃) δ: 7.39-7.36 (m, 2H), 6.81-6.78 (m, 2H),4.05-4.03 (m, 2H), 3.95 (t, J=4.2 Hz, 2H), 2.18 (bs, 1H).

Compound 2

To a solution of the compound 1 (0.3 g, 1.38 mmol) in 1,4-dioxane (10mL) at room temperature were added bis(pinacolato)diboron (0.35 g, 1.38mmol), potassium acetate (0.41 g, 4.14 mmol), and PdCl₂(dppf) (56 mg,0.07 mmol). The resulting mixture was stirred at 70° C. for 12 hours,and then concentrated under reduced pressure. Filtration was performedwith ethyl acetate. The filtered solution was concentrated under reducedpressure. The residue was subjected to column chromatography with ethylacetate and hexane to give the compound 2 (0.36 g, 97%).

¹H NMR (600 MHz, CDCl₃) δ: 7.76-7.75 (m, 2H), 6.92-6.91 (m, 2H), 4.11(t, J=4.2 Hz, 2H), 3.97-3.96 (m, 2H), 1.99 (bs, 1H), 1.33 (s, 12H).

Compound 3

A solution of the compound (A) (85 mg, 0.11 mmol), which was preparedaccording to the methods described in WO2006/111759, WO2010/043880 andWO2010/010347, the contents of each of these references are herebyincorporated by reference in their entirety, and the compound 2 (35 mg,0.13 mmol) in toluene (2 mL) were added sodium carbonate (17 mg, 0.16mmol), distilled water (1 mL), and ethanol (1 mL). After the resultingmixture was stirred for 5 minutes, Pd(TPP)₄ (22 mg, 0.02 mmol) wasadded. The resulting mixture was stirred for 2 hours. Ethyl acetate (10mL) and water (10 mL) were added. The thus-obtained organic layer wasconcentrated under reduced pressure. The residue was subjected to columnchromatography with ethyl acetate and hexane to give the compound 3 (79mg, 53%) as a yellow solid.

¹H NMR (600 MHz, CDCl₃) δ: 7.36-7.35 (m, 2H), 7.32-7.25 (m, 6H),6.92-6.89 (m, 4H), 6.78 (s, 2H), 5.92 (d, J=9.0 Hz, 2H), 5.22 (d, J=12.0Hz, 2H), 4.30-4.28 (m, 2H), 4.17-4.10 (m, 6H), 3.98-3.94 (m, 4H), 3.94(s, 6H), 3.83 (s, 3H), 3.37-3.32 (m, 2H), 2.85-2.82 (m, 2H), 2.46-2.44(m, 2H), 1.98 (bs, 1H), 0.91 (s, 18H), 0.26 (2s, 12H).

EI-MS m/z: 1387(M⁺)

Compound 4

To a solution of the compound 3 (77 mg, 0.06 mmol) in tetrahydrofuran (2mL) at room temperature were sequentially added triphenylphosphine (18mg, 0.07 mmol), phthalimide (10 mg, 0.07 mmol), and DIAD (13 ul, 0.07mmol). The resulting mixture was stirred for 12 hours. Ethyl acetate (10mL) and water (10 mL) were added. The thus-obtained organic layer wasconcentrated under reduced pressure. The residue was subjected to columnchromatography with ethyl acetate and hexane to give the compound 4 (72mg, 87%) as a yellow solid.

¹H NMR (600 MHz, CDCl₃) δ: 7.88-7.86 (m, 2H), 7.77-7.75 (m, 2H),7.39-7.36 (m, 2H), 7.30-7.24 (m, 6H), 6.90-6.86 (m, 4H), 6.78 (d, J=1.8Hz, 2H), 5.92-5.88 (m, 2H), 5.24-5.22 (m, 2H), 4.28-4.24 (m, 4H),4.17-4.11 (m, 6H), 3.98-3.90 (m, 8H), 3.83 (s, 3H), 3.36-3.29 (m, 2H),2.85-2.78 (m, 2H), 2.47-2.43 (m, 2H), 0.91 (d, J=1.8 Hz, 18H), 0.27-0.24(m, 12H).

EI-MS m/z: 1516(M⁺)

Compound 5

A solution of the compound 4 (70 mg, 0.05 mmol) in ethanol (2 mL) atroom temperature was treated with hydrazine monohydrate (12 ul, 0.23mmol). The resulting mixture was stirred at 60° C. for 5 hours. Thesolid was filtered off by using ethyl acetate (10 mL). The filtrate wasconcentrated under reduced pressure. The residue was subjected to columnchromatography with dichloromethane and methanol to give the compound 5(64 mg, 63%).

¹H NMR (600 MHz, CDCl₃) δ: 7.36-7.35 (m, 2H), 7.32-7.25 (m, 6H),6.92-6.89 (m, 4H), 6.78 (s, 2H), 5.92 (d, J=9.0 Hz, 2H), 5.22 (d, J=12.0Hz, 2H), 4.30-4.28 (m, 2H), 4.17-4.10 (m, 6H), 3.98-3.94 (m, 4H), 3.94(s, 6H), 3.83 (s, 3H), 3.37-3.32 (m, 2H), 2.85-2.82 (m, 2H), 2.46-2.44(m, 2H), 1.98 (bs, 1H), 0.91 (s, 18H), 0.26 (2s, 12H).

EI-MS m/z: 1386(M⁺)

LCB14-0599 To a solution of the compound 5 (30 mg, 0.02 mmol) intetrahydrofuran (2 mL) at room temperature were added 1N ammoniumacetate solution (0.6 mL) and cadmium/lead couple (60 mg). The resultingmixture was stirred for 4 hours. Solid was filtered off by using ethylacetate (10 mL). The filtrate was concentrated under reduced pressure.The residue was subjected to column chromatography with dichloromethaneand methanol to give the compound LCB14-0599 (9.0 mg, 60%) as a yellowsolid.

¹H NMR (600 MHz, CDCl₃, CD3OD_1drop) δ: 7.54-7.49 (m, 3H), 7.35-7.30 (m,5H), 7.26 (s, 1H), 6.93-6.86 (m, 5H), 6.51 (s, 1H), 6.29 (s, 1H),4.67-4.59 (m, 2H), 4.28-4.09 (m, 6H), 3.85 (s, 9H), 3.31-3.27 (m, 1H),3.07-3.03 (m, 2H), 2.92-2.89 (m, 1H), 2.39-2.30 (m, 2H), 2.05-2.03 (m,2H).

EI-MS m/z: 770(M⁺)

2-21. Modified GPP Derivative Including Carbonyl Group (LCB14-0606)

Compound 2

To a solution of the compound 1 (3 g, 19.45 mmol) in pyridine at roomtemperature were added acetic anhydride (7.9 mL, 77.8 mmol). Theresulting mixture was stirred for 2 hours. Petroleum ether (100 mL) and0.1N HCl (100 mL) were added. The thus-obtained organic layer wasconcentrated under reduced pressure to give the compound 2(3.81 g, 100%)in aqueous form.

¹H NMR (600 MHz, CDCl₃) δ 5.35-5.33 (m, 1H), 5.08-4.58 (m, 1H), 4.59 (d,J=6.6 Hz, 2H), 2.11-2.03 (m, 4H), 2.05 (s, 3H), 1.70 (s, 3H), 1.68 (s,3H), 1.60 (s, 3H)

Compound 3

To a solution of the compound 2 (3.81 g, 19.41 mml) in dichloromethane(30 mL) at room temperature were sequentially added selenium dioxide (65mg, 0.58 mml) and 70% tert-butylhydroperoxide (6.72 mL, 48.52 mmol). Theresulting mixture was stirred for 20 hours. After the reaction wascompleted, dichloromethane (100 mL) and water (100 mL) were added. Thethus-obtained organic layer was concentrated under reduced pressure. Theresidue was subjected to column chromatography with ethyl acetate andhexane to give the compound 3 (1.8 g, 43%) as liquid.

¹H NMR (600 MHz, CDCl₃) δ 5.38-5.30 (m, 2H), 4.59 (d, J=7.2 Hz, 2H),4.00-3.99 (d, J=6 Hz, 2H), 2.18-2.15 (m, 2H), 2.10-2.06 (m, 2H), 2.05(s, 3H), 1.70 (s, 3H), 1.66 (s, 3H)

Compound 4

To a solution of the compound 3 (1.8 g, 8.48 mmol) in dichloromethane(18 mL) at 0° C. were added triphenylphosphine (3.33 g, 12.72 mmol) andcarbon tetrabromide (3.37 g, 10.18 mmol). The resulting mixture wasstirred at 0° C. for 4 hours. Dichloromethane (100 mL) and water (100mL) were added. The thus-obtained organic layer was concentrated underreduced pressure. The residue was subjected to column chromatographywith ethyl acetate and hexane to give the compound 4 (2.33 g, 100%) inliquid form.

¹H NMR (600 MHz, CDCl₃) δ 5.57-5.55 (m, 1H), 5.35-5.32 (m, 2H), 4.59 (d,J=7.2 Hz, 2H), 3.96 (s, 2H), 2.18-2.15 (m, 2H), 2.10-2.07 (m, 2H), 2.05(s, 3H), 1.75 (s, 3H), 1.70 (s, 3H)

Compound 5

To a solution of the sodium hydride (348 mg, 8.71 mmol) intetrahydrofuran (35 mL) at 0° C. was added drop-wise a solution of ethylacetoacetate (1.85 mL, 14.52 mmol) in tetrahydrofuran (5 mL). After theresulting mixture was stirred at 0° C. for 30 minutes, the compound 4 (2g, 7.26 mmol) dissolved in tetrahydrofuran (5 mL) was slowly added at 0°C. The resulting mixture was stirred at 80° C. for 4 hours. Ethylacetate (80 mL) and water (80 mL) were added. The thus-obtained organiclayer was concentrated under reduced pressure. The residue was subjectedto column chromatography with ethyl acetate and hexane to give thecompound 5 (1.56 g, 66%) as a white liquid.

¹H NMR (600 MHz, CDCl₃) δ 5.34-5.31 (m, 1H), 5.17-5.14 (m, 1H),4.60-4.58 (m, 2H), 4.20-4.16 (m, 2H), 3.61 (t, J=7.2 Hz, 2H), 2.55-2.51(m, 2H), 2.22 (s, 3H), 2.12-2.02 (m, 4H), 2.06 (s, 3H), 1.27 (t, J=7.2Hz, 3H)

Compound 6

To a solution of the compound 5(1.56 g, 4.81 mmol) in ethanol (20 mL)was added potassium hydroxide (2.16 g, 38.47 mmol) with ethanol (20 mL).The resulting mixture was stirred 100° C. for 4 hours, diluted withethyl ether (100 mL) and 0.1N HCl solution (50 mL), and then neutralizedwith Na₂CO₃ solution. The thus-obtained organic layer was concentratedunder reduced pressure. The residue was subjected to columnchromatography to give the compound 6 (819 mg, 81%).

¹H NMR (600 MHz, CDCl₃) δ 5.39-5.37 (m, 1H), 5.09-5.07 (m, 1H), 4.15 (d,J=6.6 Hz, 2H), 2.53-2.51 (m, 2H), 2.27-2.24 (m, 2H), 2.13 (s, 3H),2.12-2.09 (m, 2H), 2.04-2.01 (m, 2H), 1.66 (s, 3H), 1.60 (s, 3H)

Compound 7

To a solution of N-chlorosuccinimide (210 mg, 1.57 mmol) indichloromethane (10 mL) under nitrogen atmosphere was slowly addeddimethyl sulfide (126 μL, 1.71 mmol). The resulting mixture was stirredat 0° C. for 5 minutes. A solution of the compound 6 (300 mg, 1.43 mmol)dissolved in dichloromethane (5 mL) was added at 30° C. The resultingmixture was stirred at 0° C. for 2 hours. After the reaction wascompleted, n-pentane (100 mL) and water (100 mL) were added. Thethus-obtained organic layer was concentrated under reduced pressure togive the compound 7 (325 mg, 99%).

¹H NMR (600 MHz, CDCl₃) δ 5.42 (m, 2H), 5.09 (m, 2H), 4.11 (d, J=8.4 Hz,2H), 2.52 (m, 2H), 2.24 (m, 2H), 2.14 (s, 3H), 2.11 (m, 2H), 2.05 (m,2H), 1.71 (s, 3H), 1.60 (s, 3H).

LCB14-0606

The compound LCB14-0606 was prepared according to the similar methoddescribed in JACS, 2010, 132(12), 4281, the contents of which areincorporated herein by reference in their entirety. To a solution of thecompound 7 (320 mg, 1.40 mmol) in 7 mL of acetonitrile at roomtemperature was slowly added a solution of tris(tetrabutylammonium)hydrogen pyrophosphate (2.25 g, 2.80 mmol) in acetonitrile (7 ml). Theresulting mixture was stirred for 1 hour. After the reaction wascompleted, the resulting mixture was concentrated under reduced pressurebelow at 25° C. The residue was subjected to column chromatography(packed BioRad AG 50W-X8 resin, hydrogen form, 15 g) with ammonia water:diluted water (V:V=3:1) and 25 mM ammonium bicarbonate:isopropyl alcohol(V:V=50:1) to give the compound LCB14-0606 (585 mg, 99%).

¹H NMR (600 MHz, D₂O) δ 5.42 (m, 1H), 5.16 (m, 1H), 4.46 (t, J=6.6 Hz,2H), 2.66 (t, J=7.2 Hz, 2H), 2.25 (t, J=7.2 Hz, 2H), 2.19 (s, 3H), 2.14(m, 2H), 2.06 (m, 2H), 1.69 (s, 3H), 1.60 (s, 3H)

Example 3: Prenylation of Ab(M)-CAAX

3-1. Prenylation methods

Prenylation of Ab(M)-CAAX was performed using NBD-GPP(Tris-ammonium[3,7-dimethyl-8-(7-nitro-benzo[1,2,5]oxadiazol-4-ylamino)-octa-2,6-diene-1]pyrophosphate)and FTase (#344146, Calbiochem, USA) or NBD-FPP (#LI-013, JenaBioscience, Germany) and GGTase I (#345852, Calbiochem, USA).

The prenylation reaction was conducted at 30° C. for 3 hours by using a50 mM Tris-HCl (pH 7.4) buffer solution containing 5 mM MgCl₂, 10 μMZnCl₂, and 5 mM DTT. After the reaction was completed, SDS-PAGE analysiswas made. An image analyzer (ChemiDoc XRS⁺, BioRad, USA) was used toidentify fluorescent protein band(s) to confirm that the prenylationreaction occurred.

3-2. Prenylation of Herceptin-HC-CAAX Using FTase and NBD-GPP

Herceptin-HC-GCVIM (“Herceptin-HC-GCVIM” disclosed as SEQ ID NO: 8),Herceptin-HC-G₅CVIM (“Herceptin-HC-G₅CVIM” disclosed as SEQ ID NO: 12)(not shown), Herceptin-HC-G₇CVIM (“Herceptin-HC-G₇CVIM” disclosed as SEQID NO: 16), and Herceptin-HC-G₁₀CVIM (“Herceptin-HC-G₁₀CVIM” disclosedas SEQ ID NO: 20) antibodies were prenylated using NBD-GPP and FTase inthe method described above. Fluorescence was detected on protein band(s)corresponding to the heavy chain(s) (about 50K dalton) of the respectiveantibodies. This result confirmed that Herceptin-HC-CAAX antibodies,each having a spacer with various lengths, could be prenylated (FIG.12).

3-3. Prenylation of Herceptin-LC-CAAX Using FTase and NBD-GPP

Herceptin-LC-GCVIM (“Herceptin-LC-GCVIM” disclosed as SEQ ID NO: 11),Herceptin-LC-G₅CVIM (“Herceptin-LC-G₅CVIM” disclosed as SEQ ID NO: 15),Herceptin-LC-G₇CVIM (“Herceptin-LC-G₇CVIM” disclosed as SEQ ID NO: 19),and Herceptin-LC-G₁₀CVIM (“Herceptin-LC-G₁₀CVIM” disclosed as SEQ ID NO:23) antibodies were prenylated using NBD-GPP and FTase in the methoddescribed above. Fluorescence was detected on protein band(s)corresponding to the light chain(s) (about 25K dalton) of the respectiveantibodies. This result confirmed that Herceptin-LC-CAAX antibodies,each having a spacer with various lengths, could be prenylated (FIG.13).

3-4. Prenylation of Anti cMET-HC-CAAX Using FTase and NBD-GPP

Anti cMET-HC-G₇CVIM (“G₇CVIM” disclosed as SEQ ID NO: 3) and anticMET-HC-G₁₀CVIM (“G₁₀CVIM” disclosed as SEQ ID NO: 4) antibodies wereprenylated using NBD-GPP and FTase in the method described above.Fluorescence was detected on protein band(s) corresponding to the heavychain(s) (about 50K dalton) of the respective antibodies. This resultconfirmed that anti cMET-HC-CAAX antibodies, each having a spacer withvarious lengths, could be prenylated (FIG. 14).

3-5. Prenylation of Anti cMET-LC-CAAX Using FTase and NBD-GPP

Anti cMET-LC-G₇CVIM (“G₇CVIM” disclosed as SEQ ID NO: 3) and anticMET-LC-G₁₀CVIM (“G₁₀CVIM” disclosed as SEQ ID NO: 4) antibodies wereprenylated using NBD-GPP and FTase in the method described above.Fluorescence was detected on protein band(s) corresponding to the lightchain(s) (about 25K dalton) of the respective antibodies. This resultconfirmed that anti cMET-LC-CAAX antibodies, each having a spacer withvarious lengths, could be prenylated (FIG. 15).

3-6. Prenylation of Herceptin-HC-CAAX Using GGTase I and NBD-FPP

A Herceptin-HC-G₁₀CVLL (“Herceptin-HC-G₁₀CVLL” disclosed as SEQ ID NO:24) antibody was prenylated using NBD-FPP and GGTase I in the methoddescribed above. Fluorescence was detected on a protein bandcorresponding to the heavy chain(s) (about 50K dalton) of the antibodythat is connected with the CAAX-motif at the C-terminus via the G₁₀ (SEQID NO: 7) spacer. This result confirmed that Herceptin-HC-CAAXantibodies could be prenylated by GGTase I (FIG. 16).

3-7. Prenylation of Herceptin-LC-CAAX Using GGTase I and NBD-FPP

A Herceptin-LC-G₁₀CVLL (“Herceptin-LC-G₁₀CVLL” disclosed as SEQ ID NO:27) antibody was prenylated using NBD-FPP and GGTase I in the methoddescribed above. Fluorescence was detected on a protein bandcorresponding to the light chain(s) (about 25K dalton) of the antibodythat is connected with the CAAX-motif at the C-terminus via the G₁₀ (SEQID NO: 7) spacer. This result confirmed that Herceptin-LC-CAAXantibodies could be prenylated by GGTase I (FIG. 16).

3-8. Prenylation of Herceptin-LC-CAAX Using FTase and Isosubstrate

Herceptin-LC-G₇CVIM (“Herceptin-LC-G₇CVIM” disclosed as SEQ ID NO: 19)

A Herceptin-LC-G₇CVIM (“Herceptin-LC-G₇CVIM” disclosed as SEQ ID NO: 19)antibody was prenylated using LCB14-0512 and FTase in the methoddescribed above. In case where the prenylated Herceptin-LC-G₇CVIM(“Herceptin-LC-G₇CVIM” disclosed as SEQ ID NO: 19) antibody wassubjected to LC/MS analysis in a reduction condition without treatingPNGase F, it was predicted that the theoretical molecular weights of theheavy chain and the light chain would be 50,597 daltons and 24,480daltons, respectively. As shown in FIG. 17, the experimental molecularweights of the heavy chain and the light chain were measured to be50,600 daltons and 24,479 daltons, respectively. The difference betweenthe theoretical molecular weight values and the experimental molecularweight values was within a standard error range. This result confirmedthat the Herceptin-LC-G₇CVIM (“Herceptin-LC-G₇CVIM” disclosed as SEQ IDNO: 19) antibody was prenylated by FTase and an isosubstrate(LCB14-0512).

Herceptin-LC-G₁₀CVIM (“Herceptin-LC-G₁₀CVIM” disclosed as SEQ ID NO: 23)A Herceptin-LC-G₁₀CVIM (“Herceptin-LC-G₁₀CVIM” disclosed as SEQ ID NO:23) antibody was prenylated using LCB14-0512 and FTase in the methoddescribed above. In the case where the prenylated HERCEPTIN-LC-G₁₀CVIM(“Herceptin-LC-G₁₀CVIM” disclosed as SEQ ID NO: 23) antibody wassubjected to LC/MS analysis in a reduction condition without treatingPNGase F, it was predicted that the theoretical molecular weights of theheavy chain and the light chain would be 50,596 daltons and 24,651daltons, respectively. As shown in FIG. 18, the experimental molecularweights of the heavy chain and the light chain were measured to be50,601 daltons and 24,651 daltons, respectively. The difference betweenthe theoretical molecular weight values and the experimental molecularweight values was within a standard error range. This result confirmedthat the Herceptin-LC-G₁₀CVIM (“Herceptin-LC-G₁₀CVIM” disclosed as SEQID NO: 23) antibody was prenylated by FTase and an isosubstrate(LCB14-0512).

Example 4: Drug Conjugation by Using Click Chemistry

4-1. Reoxidation of Prenylated Ab(M)-CAAX

Diafiltration was performed to remove excess reagents in the prenylatedHerceptin-LC-G₇CVIM (“Herceptin-LC-G₇CVIM” disclosed as SEQ ID NO: 19)prepared according to the above described method. The antibody wasreoxidized using CuSO₄. Diafiltration was performed to remove CuSO₄.

4-2. Drug Conjugation of Ab(M)-CAAX Using Click Chemistry andLinker-Drug

Click chemistry reaction between the reoxidized, prenylatedHerceptin-LC-G₇CVIM (“Herceptin-LC-G₇CVIM” disclosed as SEQ ID NO: 19)and the compound LCB14-0536 was performed for 10 minutes. The resultingconjugate (LCB14-0104) (FIG. 26) was subjected to LC/MS analysis. In thecase where the antibody was subjected to LC/MS analysis in a reductioncondition without treating PNGase F, it was predicted that thetheoretical molecular weights of the heavy chain and the light chainwould be 49,153 daltons and 25,410 daltons, respectively. As shown inFIG. 19, the experimental molecular weights of the heavy chain and thelight chain were measured to be 49,154 daltons and 25,408 daltons,respectively. The difference between the theoretical molecular weightvalues and the experimental molecular weight values was within astandard error range. This result confirmed that the prenylatedHerceptin-LC-G₇CVIM (“Herceptin-LC-G₇CVIM” disclosed as SEQ ID NO: 19)antibody formed a conjugate with a drug by click chemistry reaction.

4-3. Analysis of Herceptin-LC-CAAX-Drug Conjugates

The conjugate LCB14-0101 was subjected to hydrophobic interactionchromatography-high performance liquid chromatography with Ether-5PWcolumn (7.5×75 mm, 10 rpm, Tosoh Bioscience, USA). 50 mM potassiumphosphate buffer (pH 7.0) containing 1.5M ammonium sulfate was used asbuffer A and 50 mM potassium phosphate buffer (pH 7.0) containing 20%isopropyl alcohol was used as buffer B. 90% A/10% B was held for 5minutes. Elution was conducted using a linear gradient from 90% A/10% Bto 10% A/90% B for the next 30 minutes. The flow rate and temperaturewere set as 0.8 mL/min and 25° C., respectively. The detection wasfollowed at both 254 and 280 nm. Unmodified Herceptin-LC-G₇CVIM(“Herceptin-LC-G₇CVIM” disclosed as SEQ ID NO: 19) and prenylatedHerceptin-LC-G₇CVIM (“Herceptin-LC-G₇CVIM” disclosed as SEQ ID NO: 19)were used as controls. The retention times of the unmodifiedHerceptin-LC-G₇CVIM (“Herceptin-LC-G₇CVIM” disclosed as SEQ ID NO: 19),the prenylated Herceptin-LC-G₇CVIM (“Herceptin-LC-G₇CVIM” disclosed asSEQ ID NO: 19), and the conjugate LCB14-0101 were 9.6, 11.7, and 12.4minutes (FIG. 20), respectively.

Example 5: Antiproliferation of ADC

5-1. Cell Lines

Commercially available human breast cancer cell lines MCF-7 (HER2negative to normal), MDA-MB-468 (HER2 negative), and SK-BR-3 (HER2positive) were used. The cell lines were cultured according torecommended specifications provided with the commercially available celllines.

5-2. Test Samples

As an antibody, a commercially available Herceptin_antibody andHerceptin-LC-G₇CVIM (“Herceptin-LC-G₇CVIM” disclosed as SEQ ID NO: 19)were used. As a drug, LCB14-0537 (MMAF), LCB14-0508 (MMAF-OMe), andLCB14-0562 (MMAE) were used. As a protein-active agent conjugate,LCB14-0101, LCB14-0102, and LCB14-0103 (FIG. 26) were used. TheHerceptin-LC-G₇CVIM (“Herceptin-LC-G₇CVIM” disclosed as SEQ ID NO: 19)was prenylated using LCB14-0512. The prenylated Herceptin-LC-G₇CVIM(“Herceptin-LC-G₇CVIM” disclosed as SEQ ID NO: 19) was subjected toclick reaction using LCB14-0592 to conjugate β-glucuronidelinker(BG)-MMAF, thereby preparing LCB14-0101. In addition, theprenylated Herceptin-LC-G₇CVIM (“Herceptin-LC-G₇CVIM” disclosed as SEQID NO: 19) was subjected to click reaction using LCB14-0589 to conjugateVal-Cit linker(VC)-MMAF-OMe, thereby preparing LCB14-0102. Further, theprenylated Herceptin-LC-G₇CVIM (“Herceptin-LC-G₇CVIM” disclosed as SEQID NO: 19) was subjected to click reaction by using LCB14-0598 toconjugate β-glucuronide linker(BG)-MMAE, thereby preparing LCB14-0103.

5-3. Test Methods

Anti-proliferation activities of the antibodies, drugs, and conjugateswith regard to the cancer cell lines were measured. The cells wereplated in 96-well, tissue culture plates at 1×10⁴ cells per well. After24 hour incubation, the antibodies, drugs, and conjugates were added invarious concentrations. The number of viable cells after 72 hours werecounted using SRB dye. Absorbance was measured at 540 nm usingSpectraMax 190 (Molecular Devices, USA).

5-4. Test Results

LCB14-0101 (Herceptin-LC-G₇CVIM-BG-MMAF) (“G₇CVIM” disclosed as SEQ IDNO: 3)

Herceptin-LC-G₇CVIM (“Herceptin-LC-G₇CVIM” disclosed as SEQ ID NO: 19)had an IC₅₀ of 10 μg/mL or higher with MCF-7, MDA-MB-468, and SK-BR-3.LCB14-0101 (MMAF conjugate) had an IC₅₀ of 8.09 μg/mL and 4.18 μg/mLwith MCF-7 and MDA-MB-468, respectively, which expresses no or low levelof HER2, whereas it had an IC₅₀ of 0.11 μg/mL with SK-BR-3, whichoverexpresses HER2. In addition to its excellent inhibitory activity,LCB14-0101 is about 40-80 times more selective than Herceptin-LC-G₇CVIM(“Herceptin-LC-G₇CVIM” disclosed as SEQ ID NO: 19). Accordingly, it isconfirmed that LCB14-0101 has both cytotoxic drug potency and anti HER2selectivity (FIG. 21).

LCB14-0102 (Herceptin-LC-G₇CVIM-VC-MMAF-OM) (“G₇CVIM” Disclosed as SEQID NO: 3)

Herceptin-LC-G₇CVIM (“Herceptin-LC-G₇CVIM” disclosed as SEQ ID NO: 19)had an IC₅₀ of 10 μg/mL with MCF-7 and SK-BR-3. LCB14-0102 (MMAF-OMeconjugate) had an IC₅₀ of 4.38 μg/mL with MCF-7, whereas had an IC₅₀ of0.15 μg/mL with SK-BR-3. In addition to its excellent inhibitoryactivity, LCB14-0102 is about 30 times more selective thanHerceptin-LC-G₇CVIM (“Herceptin-LC-G₇CVIM” disclosed as SEQ ID NO: 19).Accordingly, it is confirmed that LCB14-0102 has both cytotoxic drugpotency and anti HER2 selectivity (FIG. 22).

LCB14-0103 (Herceptin-LC-G₇CVIM-BG-MMAE) (“G₇CVIM” Disclosed as SEQ IDNO: 3)

LCB14-0103 (MMAE conjugate) had an IC₅₀ of 7.25 μg/mL with MCF-7,whereas it had an IC₅₀ of 0.072 μg/mL with SK-BR-3. In addition to itsexcellent inhibitory activity, LCB14-0103 is about 100 times moreselective than Herceptin-LC-G₇CVIM (“Herceptin-LC-G₇CVIM” disclosed asSEQ ID NO: 19). Accordingly, it is confirmed that LCB14-0103 has bothcytotoxic drug potency and anti HER2 selectivity (FIG. 23).

OTHER EMBODIMENTS

From the foregoing description, it will be apparent that variations andmodifications may be made to the invention described herein to adopt itto various usages and conditions. Such embodiments are also within thescope of the following claims.

The recitation of a listing of elements in any definition of a variableherein includes definitions of that variable as any single element orcombination (or subcombination) of listed elements. The recitation of anembodiment herein includes that embodiment as any single embodiment orin combination with any other embodiments or portions thereof.

All patents and publications mentioned in this specification are hereinincorporated by reference to the same extent as if each independentpatent and publication was specifically and individually indicated to beincorporated by reference.

1-30. (canceled)
 31. An antibody-active agent conjugate comprising: (a)a full-length antibody comprising two immunoglobulin heavy chains andtwo immunoglobulin light chains, wherein the antibody recognizes andspecifically binds to a target through at least one antigen recognitionsite; (b) at least one amino acid motif having an amino acid sequenceCAAX, wherein C represents cysteine, A represents an aliphatic aminoacid, and X represents an amino acid that determines a substratespecificity of the isoprenoid transferase, directly or indirectly linkedto a carboxy terminus of a heavy chain or light chain of the antibody,wherein the amino acid motif is recognizable by an isoprenoidtransferase; (c) an isosubstrate directly linked to a cysteine moiety ofthe at least one amino acid motif, wherein the isosubstrate contains atleast one isoprenoid unit and is recognizable by the isoprenoidtransferase; and (d) at least one active agent, wherein the active agentis a drug, and the drug is directly or indirectly linked to theisosubstrate.
 32. The conjugate of claim 31, wherein the antibody is apolyclonal antibody, a monoclonal antibody, a multispecific antibody, abispecific antibody, a chimeric antibody, a humanized antibody, or ahuman antibody.
 33. The antibody-active agent conjugate of claim31,wherein the amino acid motif is directly or indirectly linked to thecarboxy terminus of an immunoglobulin heavy chain.
 34. Theantibody-active agent conjugate of claim 31, wherein the amino acidmotif is directly or indirectly linked to the carboxy terminus of animmunoglobulin light chain.
 35. The antibody-active agent conjugate ofclaim 31, wherein a second amino acid motif is directly or indirectlylinked to a second carboxy terminus.
 36. The antibody-active agentconjugate of claim 31, wherein the at least one amino acid motif isindirectly linked to the carboxy terminus of the heavy chain or thelight chain via a spacer group.
 37. The conjugate of claim 36, whereinthe spacer group comprises at least one amino acid.
 38. The conjugate ofclaim 36, wherein the spacer group comprises at least one glycine. 39.The antibody-active agent conjugate of claim 38, wherein the spacergroup comprises seven consecutive glycine residues.
 40. Theantibody-active agent conjugate of claim 31, wherein the isosubstrate isindirectly linked to the at least one active agent via at least onelinker.
 41. The antibody-active agent conjugate of claim 40, wherein thelinker is a linear linker.
 42. The antibody-active agent conjugate ofclaim 41, wherein the linear linker is directly linked to the at leastone active agent.
 43. The antibody-active agent conjugate of claim 40,wherein the linker is a linker having branches.
 44. The antibody-activeagent conjugate of claim 43, wherein one or more of the branches aredirectly linked to at least one of the active agents.
 45. Theantibody-active agent conjugate of claim 43, wherein at least two of thebranches are directly linked to different active agents.
 46. Theantibody-active agent conjugate of claim 40, wherein the linker is acleavable linker.
 47. The antibody-active agent conjugate of claim 46,wherein the linker is a chemically cleavable linker, an enzymaticallycleavable linker, a hydrolysable linker, or a combination thereof. 48.The antibody-active agent conjugate of claim 47, wherein the cleavablelinker is an enzymatically cleavable linker and the enzymaticallycleavable linker contains a peptide that can be cleaved by cathepsin Bor a glucuronide that can be cleaved by β-glucuronidase.
 49. Theantibody-active agent conjugate of claim 31, wherein the drug iserlotinib, bortezomib, fulvestrant, sutent, letrozole, imatinibmesylate, PTK787/ZK 222584, oxaliplatin, 5-fluorouracil, leucovorin,rapamycin, lapatinib, lonafarnib, sorafenib, gefitinib, TyrphostinAG1478, an inhibitor of EGFR tyrosine kinase activity, thiotepa,cyclophosphamide, busulfan, improsulfan, piposulfan, benzodopa,carboquone, meturedopa, uredopa; ethylenimine, altretamine,triethylenemelamine, triethylene phosphoramide, triethylenethiophosphoramide, trimethylol melamine, bullatacin, bullatacinone,camptothecin topotecan, bryostatin; callystatin; adozelesin, carzelesin,bizelesin, cryptophycin 1, cryptophycin 8; dolastatin; duocarmycin,eleutherobin; pancratistatin; sarcodictyin; spongistatin, chlorambucil,chlornaphazine, cyclophosphamide, estramustine, ifosfamide,mechlorethamine, mechlorethamine oxide hydrochloride, melphalan,novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard,carmustine, chlorozotocin, fotemustine, lomustine, nimustine,calicheamicin, calicheamicin gamma 1I, calicheamicin omega I1,dynemicin, dynemicin A, clodronate, esperamicin, neocarzinostatinchromophore, aclacinomysins, actinomycin, anthramycin, azaserine,bleomycins, cactinomycin, carabicin, carninomycin, carzinophilin,chromomycins, dactinomycin, daunorubicin, 6-diazo-5-oxo-L-norleucine,doxorubicin, morpholino-doxorubicin, cyanomorpholino-doxorubicin,2-pyrrolino-doxorubucin, liposomal doxorubicin, deoxydoxorubicin,epirubicin, esorubicin, marcellomycin, mitomycin C, mycophenolic acid,nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin,quelamycin, rodorubicin, streptomigrin, streptozocin, tubercidin,ubenimex, zinostatin, zorubicin, 5-fluorouracil, denopterin,methotrexate, pteropterin, trimetrexate, fludarabine, 6-mercaptopurine,thiamiprine, tioguanine, ancitabine, azacitidine, 6-azauridine,carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine,floxuridine, calusterone, dromostanolone propionate, epitiostanol,mepitiostane, testolactone, aminoglutethimide, mitotane, trilostane,folinic acid; aceglatone; aldophosphamide glycoside; aminolevulinicacid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate;demecolcine; diaziquone; elfornithine; elliptinium acetate, etoglucid;gallium nitrate; hydroxyurea; lentinan; lonidainine, maytansine,ansamitocins; mitoguazone; mitoxantrone; mopidanmo; nitraerine;pentostatin; phenamet; pirarubicin; losoxantrone; 2-ethylhydrazide;procarbazine, polysaccharide-k, razoxane; rhizoxin; sizofiran;spirogermanium; tenuazonic acid; triaziquone;2,2′,2″-trichlorotriethylamine, verracurin A, roridin A, anguidine;urethane; vindesine; dacarbazine; mannomustine; mitobronitol;mitolactol; pipobroman; gacytosine; arabinoside (‘Ara-C’);cyclophosphamide; thiotepa, paclitaxel, doxetaxel; chlorambucil;gemcitabine; 6-thioguanine; mercaptopurine, cisplatin, carboplatin,vinblastine, platinum, etoposide, ifosfamide, mitoxantrone, vincristine,vinorelbine; novantrone; teniposide; edatrexate; daunomycin;aminopterin; ibandronate; CPT-11; topoisomerase inhibitor RFS 2000;difluoromethylornithine (DFMO), retinoic acid; capecitabine; or apharmaceutically acceptable salt, solvate, or acid of any of theforegoing;
 50. The antibody-active agent conjugate of claim 31, whereinthe amino acid motif is CVIM (SEQ ID NO:28) or CVLL (SEQ ID NO:29). 51.The antibody-active agent conjugate of claim 31, wherein the drug isdirectly or indirectly linked to the isosubstrate by an oxime.
 52. Theantibody-active agent conjugate of claim 40, wherein the linker is anon-cleavable linker.
 53. The antibody-active agent conjugate of claim41, wherein the linear linker is directly linked to the at least twodifferent active agents.